Devices and methods for isolating tumor infiltrating lymphocytes and uses thereof

ABSTRACT

The present invention provides methods for isolating and cryopreserving tumor infiltrating lymphocytes (TILs) and producing therapeutic populations of TILs, including methods via use of a kit and a semi-automatic device for aseptic disaggregation, enrichment, and cryopreservation of a resected tumor prior to expansion of the TIL population. The present invention also provides methods for expansion, and/or stabilization of TILs, for instance UTILs, compositions involving the same and methods of treatment involving the same.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation U.S. patent application Ser. No.17/733,875, filed Apr. 29, 2022, which is a continuation ofPCT/GB2020/053315 filed Dec. 18, 2020, which claims the benefit ofpriority from U.S. Patent Application Ser. No. 62/951,559 filed Dec. 20,2019, U.S. Patent Application Ser. No. 62/982,470 filed Feb. 27, 2020,and U.S. Patent Application Ser. No. 63/047,431 filed Jul. 2, 2020, thecontents of which are incorporated herein by reference in theirentireties.

Reference is made to United Kingdom patent application Serial No.GB1700621.4, filed Jan. 13, 2017, European patent applicationEP18701791.8, filed Jan. 12, 2018, international patent applicationSerial No. PCT/GB2018/050088, filed Jan. 12, 2018, published as PCTPublication No. WO 2018/130845 on Jul. 19, 2018, European patentpublication: EP3568459, and U.S. Patent Application Ser. No. 62/951,559,filed Dec. 20, 2019, which are hereby incorporated reference.

Reference is made to United Kingdom patent application Serial No.GB1902763.0, filed Mar. 1, 2019, United Kingdom patent applicationSerial No. GB1904249.8, filed Mar. 27, 2019, and international patentapplication Serial No. PCT/EP2020/000053, filed Feb. 28, 2020, publishedas WO 2020/177920 on Sep. 10, 2020.

The foregoing applications, Biomarker Predictive of Tumour InfiltratingLymphocyte Therapy and the Uses Thereof, WO2019145711A1 PCT/GB2019/050188, Tumor Infiltrating Lymphocyte Therapy and Uses Thereof USA,PCT/GB2020/051790 and U.S. application Ser. No. 62/878,001, ReceptorsProviding Targeted Costimulation for Adoptive Cell Therapy WO2020/152451, U.S. application Ser. No. 62/951,770 and GB1900858.0, CellsExpressing Recombinant Growth Factor Receptors WO 2017/103596A1, U.S.application Ser. No. 16/061,435, and European patent publicationEP3390436, and Chimeric Growth Factor Receptors WO2019243835A1PCT/GB2019/051745, and all documents cited therein or during theirprosecution (“appin cited documents”) and all documents cited orreferenced in the appin cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention provides methods and devices for isolating andfreezing tumor infiltrating lymphocytes (TILs) from a resected tumor viasemi-automatic aseptic tissue processing of the tumor and therebyproducing therapeutic populations of TILs.

BACKGROUND OF THE INVENTION

T cells are derived from hemopoietic stem cells resident in bone marrowbut subsequently migrate to and mature in the thymus. During the processof maturation, T cells undergo a series of selection events, therebygenerating a diverse repertoire of T cells. These cells are thenreleased into the peripheral circulation to carry out their specificfunctions as a part of the adaptive immune system.

T cells are not a homogeneous group of cells but consist of manylineages, of which the predominant types are defined by the expressionof two further cell markers. CD4 expressing T cells are generally termedhelper (Th) and are thought to orchestrate many functions of the immunesystem by cell-cell contact and through the production of mediatormolecules called cytokines. CD8 T cells are considered to be cytotoxic(Tc) and are thought to be the cells which perform direct killing oftarget cells. These activities are all controlled through the T cellreceptor/antigen/MHC interaction—consequently, upon successfulrecognition of a peptide/MHC on a target cell, CD4 and CD8 cells act inconcert through cytokine production and cytotoxic activity to eliminatetarget cells, including virus infected and tumor cells.

T cells do not recognize intact proteins (antigens) but respond toshort, protein fragments presented on the surface of target cells byspecific proteins called the Major Histocompatibility Complex (MHC).During the maturation process, T cells express on their cell surface anantigen-specific T cell receptor (TCR), which recognizes these shortprotein (peptide) antigens presented by MHC molecules. Consequently,only when the correct peptide is presented on the surface of a targetcell associated with the correct MHC molecule will the T cell activateits immune functions. Therefore, the frequency of tumor specific T cellsare enriched in the tumor making it an ideal source for tumor specific Tcells i.e. tumor-infiltrating lymphocytes (TIL) (Andersen et al., CancerRes. 2012 Apr. 1; 72(7):1642-50. doi: 10.1158/0008-5472.CAN-11-2614.Epub 2012 Feb. 6).

Of course, this is a highly simplified view and represents a shortgeneral overview of T cell function. The adaptive immune response doesnot act in isolation but requires extensive interaction with a range ofimmune and non-immune cells to facilitate the efficient trafficking of Tcells to the required site of activity, to ensure that the correctimmune response is initiated and that the immune response is controlledand turned off after it is needed. Therefore, even in patients where themanufactured TIL initiate an immune response to the tumor it may then besupported or dampened by the patient's own immune system and the tumorenvironment.

Tumor specific TIL are T cells isolated from a tumor of a patient withmetastatic cancer. In most cancer patients circulating tumor-specific Tcells can hardly be detected in blood. However, certain cancers such ascutaneous melanoma appear to be immunogenic as it has the ability toinduce significant numbers of T cells with anti-tumor activity duringthe natural course of the tumor growth, especially within the tumorareas (Muul et al., J Immunol. 1987 Feb. 1:138(3):989-95).Tumor-reactive T cells “selected as T cell specific for the tumor” canbe isolated from tumor material and expanded ex vivo into high numbers.Reports have shown that these cells contain anti-tumor reactivity, whichcan result in tumor destruction and clinical responses upon reinfusioninto the patient (Dudley et al., Science. 2002 Oct. 25; 298(5594):850-4.Epub 2002 Sep. 19). In subsequent trials the importance of T cellcharacteristics was confirmed and the benefit of “young” rapidly growingcells “Young TILs” was confirmed whereby cells are “not selected forspecificity” at all. Remarkably this produces excellent response ratesin TIL or CD8 selected TIL of around 50% (Besser et al., Anticancer Res.2009 January:29(1):145-54; Dudley et al., Clin Cancer Res. 2010 Dec. 15;16(24):6122-31. doi: 10.1158/1078-0432.CCR-10-1297. Epub 2010 Jul. 28).

Studies by Andersen et al. (Cancer Res. 2012 Apr. 1; 72(7):1642-50. doi:10.1158/0008-5472.CAN-11-2614. Epub 2012 Feb. 6) identified thatmelanoma specific T cells (for known cancer antigens) are enrichedwithin the tumor compared with T cells in the peripheral blood. Thissupports the dogma that the isolated TIL population are enriched tumorspecific T cells resulting in an enhanced anti-tumor activity whencompared with early trials in melanoma patients using T cells isolatedfrom peripheral blood and expanded in similar levels of IL2 orintravenous IL-2 alone (LAK cells—Bordignon et al., Haematologica. 1999December; 84(12):1110-49).

U.S. Pat. No. 10,398,734 relates to methods for expanding TILs andproducing therapeutic populations of TILs. The tumor of the '734 patentis shipped as a bulk tumor, and the TILs inside the bulk tumor rapidlybecome oxygen deficient and deteriorate progressively over time. Thetumor of the '734 patent is also processed to fragments which havedeteriorated internal cell populations. Furthermore, the TILs used formanufacturing will only be TILs expanded from tissue fragments and notany TILs retained in the interior. Therefore, the resulting cellpopulation may not reflect the full diversity of the tumor environment.

Harvesting TILs requires the aseptic disaggregation of solid tissue as abulk tumor prior to the culture and expansion of the TIL population. Theconditions during solid tissue disaggregation and time taken to harvestthe cells have a substantial impact on the viability and recovery of thefin al cellularized material. A solid tissue derived cell suspensionthat is obtained using conventional methods often includes a widevariety of different cell types, disaggregation media, tissue debrisand/or fluids. This may necessitate the use of selective targetingand/or isolation of cell types, for example, prior to manufacture ofregenerative medicines, adoptive cell therapies, ATMPs, diagnostic invitro studies and/or scientific research.

Currently, selection or enrichment techniques generally utilize one of:size, shape, density, adherence, strong protein-protein interactions(i.e. antibody-antigen interactions). For example, in some instancesselection may be conducted by providing a growth supporting environmentand by controlling the culture conditions or more complex cell markerinteractions associated with semi-permanent or permanent coupling tomagnetic or non-magnetic solid or semisolid phase substrates.

For enrichment, isolation, or selection, any sorting technology can beused, for example, affinity chromatography or any otherantibody-dependent separation technique known in the art. Anyligand-dependent separation technique known in the art may be used inconjunction with both positive and negative separation techniques thatrely on the physical properties of the cells. An especially potentsorting technology is magnetic cell sorting. Methods to separate cellsmagnetically are commercially available e.g. from Thermo Fisher,Miltenyi Biotech, Stemcell Technologies, Cellpro Seattle, AdvancedMagnetics, Boston Scientific, or Quad Technologies. For example,monoclonal antibodies can be directly coupled to magnetic polystyreneparticles like Dynal M 450 or similar magnetic particles and used, forexample for cell separation. The Dynabeads technology is not columnbased, instead these magnetic beads with attached cells enjoy liquidphase kinetics in a sample tube, and the cells are isolated by placingthe tube on a magnetic rack.

Enriching, sorting and/or detecting cells from a sample includes usingmonoclonal antibodies in conjunction with colloidal superparamagneticmicroparticles having an organic coating of, for example,polysaccharides (e.g. magnetic-activated cell sorting (MACS) technology(Miltenyi Biotec, Bergisch Gladbach, Germany)). Particles (e.g.,nanobeads or MicroBeads) can be either directly conjugated to monoclonalantibodies or used in combination with anti-immunoglobulin, avidin, orantihapten-specific MicroBeads, or coated with other mammalian moleculeswith selective binding properties.

Magnetic particle selection technologies such as those described above,allows cells to be positively or negatively separated by incubating themwith magnetic nanoparticles coated with antibodies or other moietiesdirected against a particular surface marker. This causes the cellsexpressing this marker to attach to the magnetic nanoparticles.Afterwards the cell solution is placed within a solid or flexiblecontainer in a strong magnetic field. In this step, the cells attach tothe nanoparticles (expressing the marker) and stay on the column, whileother cells (not expressing the marker) flow through. With this method,the cells can be separated positively or negatively with respect to theparticular marker(s).

In case of a positive selection the cells expressing the marker(s) ofinterest, which attached to the magnetic column, are washed out to aseparate vessel, after removing the column from the magnetic field.

In case of a negative selection the antibody or selective moiety used isdirected against surface markers(s) which are known to be present oncells that are not of interest. After application of the cells/magneticnanoparticles solution onto the column the cells expressing theseantigens bind to the column and the fraction that goes through iscollected, as it contains the cells of interest. As these cells arenon-labelled by the selective antibodies or moiety(s) coupled tonanoparticles, they are “untouched”. The known manual or semi-automatedsolid tissue processing steps are labor-intensive and require aknowledge of the art.

In addition, where the material is used for therapeutic purposes, theprocessing requires strict regulated environmental conditions duringhandling of the cell cultures, for example tissue processing as a partof or prior to disaggregation, enzymatic digestion and transfer intostoring devices, or incubation conditions fordisaggregation/cellularization and viable tissue yields. Typically, thisprocess would require multiple pieces of laboratory and tissueprocessing equipment, and personnel with the skills and knowledge of thescientific art with critical stages contained within either hazardcontainment or tissue processing facility(s) aseptic environment(s) inorder to perform the same activity safely and also minimize the risk ofcontamination(s).

Viability and recovery of a desired product from tissue may be affectedby the conditions during tissue collection, disaggregation, andharvesting of cells. The invention arises from a need to provideimproved tissue processing, including an apparatus/device thatundertakes said processing that achieves the unmet need described above.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention relates to a method for isolating a therapeuticpopulation of tumor infiltrating lymphocytes (TIL) which may comprise:

-   -   (a) resecting a tumor from a subject;    -   (b) storing the resected tumor in a single use aseptic kit,        wherein the aseptic kit comprises:        -   a disaggregation module for receipt and processing of            material comprising solid mammalian tissue;        -   an optional enrichment module for filtration of            disaggregated solid tissue material and segregation of            non-disaggregated tissue and filtrate; and a stabilization            module for optionally further processing and/or storing            disaggregated product material,        -   wherein each of the modules comprises one or more flexible            containers connected by one or more conduits adapted to            enable flow of the tissue material there between; and        -   wherein each of the modules comprises one or more ports to            permit aseptic input of media and/or reagents into the one            or more flexible containers;    -   (c) aseptically disaggregating the resected tumor in the        disaggregation module thereby producing a disaggregated tumor;    -   (d) performing a first expansion by culturing the disaggregated        tumor in a cell culture medium comprising IL-2 to produce a        first population of UTILs;    -   (e) performing a second expansion by culturing the first        population of UTILs with additional 1L-2, OKT-3, and antigen        presenting cells (APCs), to produce a second population of TILs;    -   (f) harvesting and/or cryopreserving the second population of        UTILs. In some embodiments, step a) is optional.

The present invention relates to a method for isolating a therapeuticpopulation of cryopreserved tumor infiltrating lymphocytes (TIL) whichmay comprise:

-   -   (a) resecting a tumor from a subject;    -   (b) storing the resected tumor in a single use aseptic kit,        wherein the aseptic kit comprises:        -   a disaggregation module for receipt and processing of            material comprising solid mammalian tissue;        -   an optional enrichment module for filtration of            disaggregated solid tissue material and segregation of            non-disaggregated tissue and filtrate; and a stabilization            module for optionally further processing and/or storing            disaggregated product material,        -   wherein each of the modules comprises one or more flexible            containers connected by one or more conduits adapted to            enable flow of the tissue material there between; and        -   wherein each of the modules comprises one or more ports to            permit aseptic input of media and/or reagents into the one            or more flexible containers;    -   (c) aseptically disaggregating the resected tumor in the        disaggregation module thereby producing a disaggregated tumor,        wherein the resected tumor is sufficiently disaggregated if it        can be cryopreserved with a minimum of cell damage;    -   (d) cryopreserving the disaggregated tumor in the stabilization        module;    -   (e) performing a first expansion by culturing the disaggregated        tumor in a cell culture medium comprising IL-2 to produce a        first population of UTILs;    -   (f) performing a second expansion by culturing the first        population of UTILs with additional IL-2, OKT-3, and antigen        presenting cells (APCs), to produce a second population of TILs;    -   (g) harvesting and/or cryopreserving the second population of        UTILs. In some embodiments, step a) is optional.

The disaggregation may comprise physical disaggregation, enzymaticdisaggregation, or physical and enzymatic disaggregation. In anadvantageous embodiment, the disaggregated tumor is cellularized orpurified.

In the present invention, sets of containers, which are interconnectedand have specific separate functions maintain an aseptically closedsystem to process, optionally enrich but stabilize the disaggregated andcellularized tumor. Essentially the invention provides a rapidpre-sterilized environment to minimize the time required and risk ofcontamination or operator exposure during the processing of the resectedtumor.

The aseptic kit allows for closed solid tissue processing, eliminatingthe risk of contamination of the final cellularized product compared tostandard non-closed tissue processing, especially when the process isperformed within a tissue retrieval/procurement site and requiresstorage prior to final cell processing for its ultimate utility. Inaddition, safety of the operator is increased due to reduction of directcontact with biological hazardous material, which may contain infectiousorganisms such as viruses. The kit also enables either all of or aportion of the finally processed cellularized material to be stabilizedfor either transport or storage prior to being processed for itsultimate utility.

The invention will enable the resected tumor to be processed at the timeof resection, or later if required, without impact upon the retrievalprocedure or the viability of the cellularized tumor.

In some embodiments, an optional enrichment via a form of physicalpurification to reduce impurities such as no longer required reagents;cell debris; non-disaggregated tumor tissue and fats can be employed.The aseptic kit can have an optional enrichment module, prior tostabilization, for this purpose. A single cell or small cell numberaggregates can be enriched for stabilization after disaggregation byexcluding particles and fluids of less than 5 μm or incompletelydisaggregated material of or around 200 μm across or larger but thiswill vary upon the tissue and the efficiency of disaggregation andvarious embodiments in the form of tissue specific kits may be employeddepending upon the tissue or ultimate utility of the disaggregatedtumor.

In another embodiment, a single cell suspension is provided after step(c).

In another embodiment, the first population of UTILs requires about 1-20million UTILs.

In another embodiment, step (e) may further comprise growth of the UTILsout of the resected tumor starting material followed by the rapidexpansion of step (f).

In another embodiment, step (e) may be performed for about two weeks andstep (f) may be performed for about two weeks.

In another embodiment, additional step (h) involves suspending thesecond population of UTILs. The suspending may be in buffered saline,human serum albumin, and/or dimethylsulfoxide (DMSO).

The present invention also may comprise a therapeutic population ofcryopreserved UTILs obtained by any of the herein disclosed methods. Thetherapeutic population may comprise about 5×10⁹ to 5×10¹⁰ of T cells.

The present invention also encompasses a cryopreserved hag of the hereindisclosed therapeutic population. The cryopreserved bag may be for usein intravenous infusion.

The present invention also encompasses a method for treating cancerwhich may comprise administering the herein disclosed therapeuticpopulation or the herein disclosed cryopreserved bag. The presentinvention also encompasses the herein disclosed therapeutic population,pharmaceutical composition or cryopreserved bag for use in the treatmentof cancer. The cancer may be bladder cancer, breast cancer, cancercaused by human papilloma virus, cervical cancer, head and neck cancer(including head and neck squamous cell carcinoma (HNSCC), lung cancer,melanoma, ovarian cancer, non-small-cell lung cancer (NSCLC), renalcancer, or renal cell carcinoma.

In another embodiment, the one or more flexible containers of theaseptic kit comprise a resilient deformable material.

In another embodiment, the one or more flexible containers of thedisaggregation module of the aseptic kit comprises one or more sealableopenings. The one or more flexible containers of the disaggregationmodule and/or the stabilization module may also comprise a heat sealableweld.

In another embodiment, the one or more flexible containers of theaseptic kit comprises internally rounded edges.

In another embodiment, the one or more flexible containers of thedisaggregation module of the aseptic kit comprises disaggregationsurfaces adapted to mechanically crush and shear the solid tumortherein.

In another embodiment, the one or more flexible containers of theenrichment module of the aseptic kit comprises a filter that retains aretentate of cellularized disaggregated solid tumor.

In another embodiment, the one or more flexible containers of thestabilization module of the aseptic kit comprises media formulation forstorage of viable cells in solution or in a cryopreserved state.

In another embodiment, the aseptic kit further comprises a digital,electronic, or electromagnetic tag identifier. The tag identifier canrelate to a specific program that defines a type of disaggregationand/or enrichment and/or stabilization process, one or more types ofmedia used in said processes, including an optional freezing solutionsuitable for controlled rate freezing.

In another embodiment, the same flexible container can form part of oneor more of the disaggregation module, the stabilization module, and theoptional enrichment modules.

In another embodiment, the disaggregation module of the aseptic kitcomprises a first flexible container for receipt of the tissue to beprocessed.

In another embodiment, the disaggregation module of the aseptic kitcomprises a second flexible container comprising the media fordisaggregation.

In another embodiment, the optional enrichment module of the aseptic kitcomprises the first flexible container and a third flexible containerfor receiving the enriched filtrate.

In another embodiment, both the disaggregation module and thestabilization module of the aseptic kit comprise the second flexiblecontainer and the second flexible container comprises digestion mediaand stabilization media.

In another embodiment, the stabilization module of the aseptic kitcomprises a fourth flexible container comprising stabilization media.

In another embodiment, the stabilization module of the aseptic kit alsocomprises the first flexible container and/or third flexible containerfor storing and/or undergoing cryopreservation.

The present invention also provides for a method for isolating atherapeutic population of cryopreserved UTILs comprising:

-   -   (a) resecting a tumor from a subject;    -   (b) storing the resected tumor in a single use aseptic kit,        wherein the aseptic kit comprises: a disaggregation module for        receipt and processing of material comprising solid mammalian        tissue;        -   an optional enrichment module for filtration of            disaggregated solid tissue material and segregation of            non-disaggregated tissue and filtrate; and a stabilization            module for optionally further processing and/or storing            disaggregated product material,        -   wherein each of the modules comprises one or more flexible            containers connected by one or more conduits adapted to            enable flow of the tissue material there between; and        -   wherein each of the modules comprises one or more ports to            permit aseptic input of media and/or reagents into the one            or more flexible containers;    -   (c) aseptically disaggregating the resected tumor in the        disaggregation module thereby producing a disaggregated tumor,        wherein the resected tumor is sufficiently disaggregated if it        can be cryopreserved without cell damage;    -   (d) cryopreserving the disaggregated tumor in the stabilization        module;    -   (e) performing a first expansion by culturing the disaggregated        tumor in a cell culture medium comprising IL-2 to produce a        first population of UTILs;    -   (f) performing a second expansion by culturing the first        population of UTILs with additional IL-2, OKT-3, and antigen        presenting cells (APCs), to produce a second population of TILs;    -   (g) harvesting and/or cryopreserving the second population of        UTILs. In some embodiments, step a) is optional.

In another embodiment, the automated device further comprises a radiofrequency identification tag reader for recognition of the aseptic kitso that it may be scanned and recognized during automated processing,such as within the automated device in embodiments of the presentinvention. Crucially the tag provides information about the conditionsand steps required to be auto processed, so simply by scanning the kit,any automated system used with the kit to process the tissue can beundertaken without further intervention or contamination. Once thetissue sample has been placed in the disaggregation module, it can forexample be sealed, manually or automatically, before processing begins.

The programmable processor of the automated device can also recognizethe aseptic kit via the tag and subsequently can execute the kit programdefining the type of disaggregation, enrichment, and stabilizationprocesses, and the respective media types required for said processes,which include an optional freezing solution suitable for controlled ratefreezing. The programmable processor of the automated device isadaptable to communicate with and control the disaggregation module, theenrichment module, and/or the stabilization module. Put another way, thekit is therefore readable by an automated device used to execute aspecific fully automatic method for processing the tumor when insertedinto such a device.

The programmable processor of the automated device can control thedisaggregation module to enable a physical and/or biological breakdownof the solid tissue material. This breakdown can be a physical orenzymatic breakdown of the solid tissue material. Enzymatic breakdown ofthe solid tissue material can be by one or more media enzyme solutionsselected from the group consisting of collagenase, trypsin, lipase,hyaluronidase, deoxyribonuclease, Liberase HI, pepsin, and mixturesthereof.

In another embodiment, the programmable processor controlsdisaggregation surfaces within the disaggregation flexible containersthat mechanically crush and shear the solid tissue. In some embodiments,the disaggregation surfaces are controlled by mechanical pistons.

In another embodiment, the programmable processor controls thestabilization module to cryopreserve the enriched disaggregated solidtissue in the container. This may be achieved using a programmabletemperature setting, a condition which is determined by reading the tagof the kit inserted in the device.

In another embodiment, to undertake different functions of the process,one or more of the additional components of the device and/or kit areprovided and may be available in any combination. This may include:sensors capable of recognizing whether a disaggregation process has beencompleted in the disaggregation module prior to transfer of thedisaggregated solid tissue to the optional enrichment module; weightsensors to determine an amount of media required in the containers ofone or more of the disaggregation module; the enrichment module; and/orthe stabilization module and control the transfer of material betweenrespective containers; sensors to control temperature within thecontainers of the one or more of the disaggregation module; theenrichment module; and/or the stabilization module; at least one bubblesensor to control transfer of media between the input and output portsof each container in the module; at least one pump, optionally aperistaltic pump, to control transfer of media between the input andoutput ports; pressure sensors to assess the pressure within theenrichment module; one or more valves to control a tangential flowfiltration process within the enrichment module; and/or one or moreclamps to control the transfer of media between the input and outputports of each module.

In another embodiment, the programmable processor of the automateddevice is adapted to maintain an optimal storage temperature range inthe stabilization module until the container is removed; or executes acontrolled freezing step. This allows the UTILs to be stored for shortperiods (minutes to days) or stored for long periods (multiple days toyears) prior to their ultimate utility depending on the type orstabilization process used with the stabilization module.

In another embodiment, the automated device further comprises a userinterface. The interface can comprise a display screen to displayinstructions that guide a user to input parameters, confirmpre-programmed steps, warn of errors, or combinations thereof.

In another embodiment, the automated device is adapted to betransportable and thus may comprise dimensions that permit easymaneuverability and/or aid movement such as wheels, tires, and/orhandles.

The present invention also provides a semi-automatic aseptic tissueprocessing method for isolating a therapeutic population ofcryopreserved UTILs comprising the steps of:

-   -   (a) automatically determining aseptic disaggregation tissue        processing steps and their associated conditions from a digital,        electronic, or electromagnetic tag identifier associated with an        aseptic processing kit, wherein the aseptic kit comprises:        -   a disaggregation module for receipt and processing of            material comprising solid        -   mammalian tissue;        -   an optional enrichment module for filtration of            disaggregated solid tissue material and segregation of            non-disaggregated tissue and filtrate; and a stabilization            module for optionally further processing and/or storing            disaggregated product material,        -   wherein each of the modules comprises one or more flexible            containers connected by one or more conduits adapted to            enable flow of the tissue material there between; and        -   wherein each of the modules comprises one or more ports to            permit aseptic input of media and/or reagents into the one            or more flexible containers;    -   (b) resecting a tumor from a subject;    -   (c) placing the tumor into the flexible plastic container of the        disaggregation module of the aseptic kit;    -   (d) processing the tumor by automatically executing the one or        more tissue processing steps by communicating with and        controlling:        -   the disaggregation module; wherein the resected tumor is            aseptically disaggregated thereby producing a disaggregated            tumor, wherein the resected tumor is sufficiently            disaggregated if it can be cryopreserved without cell            damage;        -   the optional enrichment module wherein the disaggregated            tumor is filtered to remove disaggregated solid tissue            material and to segregate non-disaggregated tissue and            filtrate;        -   the stabilization module wherein the disaggregated tumor is            cryopreserved;    -   (e) performing a first expansion by culturing the disaggregated        tumor in a cell culture medium comprising IL-2 to produce a        first population of UTILs;    -   (f) performing a second expansion by culturing the first        population of UTILs with additional IL-2, OKT-3, and antigen        presenting cells (APCs), to produce a second population of TILs;    -   (g) harvesting and/or cryopreserving the second population of        UTILs. In some embodiments, step b) is optional.

Flexible containers such as bags, may be used to process tissuematerials. Processing may include treatments that may separate orbreakdown tissue, for example, physical breakdown may be accomplishedusing agitation, e.g., gentle agitation, a biological and/or enzymaticbreakdown may include enzymatic digestion, and/or extraction ofcomponents of the tissue materials in the bag.

A flexible container, such as a bag, for processing tissue may includeone or more layers made of a sealable polymer having at least threeedges of the flexible container which are sealed during manufacturingand an open edge on the flexible container through which tissue materialis inserted during use. One or more connectors may be used to couple theflexible container to at least one element through tubing. After tissueis placed in the flexible container, a section of the flexible containerproximate the open edge may be sealed or welded to form a seal. The sealmay have a width of at least a three mm and be positioned substantiallyparallel to the open edge and spaced away from the open edge of theflexible container. In some instances, the seal may have a width greaterthan about five mm. For example, a bag may be sealed after tissue isplaced inside to have a seal of least 5 mm positioned proximate the openedge of the bag. The seal may be parallel to the open edge and spacedaway from the open edge of the bag.

The flexible container may be further secured using a clamp havingprotrusions and positioned proximate the seal and spaced further fromthe open edge of the flexible container than the seal.

In some instances, the seal and the flexible container are constructedsuch that the flexible container can withstand a 100 N force applied tothe flexible container during use. Using a clamp in conjunction withsuch a seal may be advantageous in some instances depending on the typeof material used and/or a structure of the seal. Thus, during use of aflexible container, such as a hag, a combination of a seal and a clampmay be capable of withstanding a 100 N force applied to the flexiblecontainer.

In some instances, the seal and the flexible container are constructedsuch that the flexible container can withstand a 75 N force applied tothe flexible container during use. Using a clamp in conjunction withsuch a seal may be advantageous in some instances depending on the typeof material used and/or a structure of the seal. Thus, during use of aflexible container, such as a bag, a combination of a seal and a clampmay be capable of withstanding a 75 N force applied to the flexiblecontainer.

A flexible container may be used to hold tissue during processing suchas disaggregation of the tissue material.

In some embodiments, a flexible container, such as a bag, may be usedfor disaggregation of the tissue material, filtration of disaggregatedtissue material, and/or segregation of non-disaggregated tissue andfiltrate.

Flexible containers such as bags may be formed from a resilientdeformable material. Materials for use in flexible containers, such asbags may be selected for one or more properties including but notlimited to sealability such as sealability due to heat welding, or useof radio frequency energy, gas permeability, flexibility for example lowtemperature flexibility (e.g., at −50° C., or −195° C.), elasticity forexample low temperature elasticity, chemical resistance, opticalclarity, biocompatibility such as cytotoxicity, hemolytic activity,resistance to leaching, having low particulates, high transmissionsrates for particular gases (e.g., Oxygen and/or Carbon dioxide), and/orcomplying with regulatory requirements.

Flexible containers, such as bags, may include indicators. Indicatorsmay be used to identify samples, patients from whom the samples werederived, and/or to track progress of a particular sample through atreatment process. In some instances, indicators may be scanned by anautomated or semi-automated system to track progress of a sample.

Marks may be used on a flexible container, such as a bag, to identifywhere the bag should be placed, treated, sealed, or any other actionthat may be taken with respect to a bag that includes tissue. Each bagmay include multiple marks for sealing.

An open end of the bag may be sealed after tissue is inserted in thebag. Any seal may be formed using a sealing device (e.g., heater sealer)operating at a predetermined pressure, a predetermined temperature, andpredetermined time frame.

In some instances, a flexible container, such as a hag may be used as adisaggregation container for use as part of a disaggregation elementthat may also include a disaggregation device. In some embodiments,media and/or enzymes may be added to the a bag within a disaggregationelement of a device. For example, a bag may be used with a device thatmechanically crushes tissue material placed in the flexible container.

In some embodiments, tissue in a flexible container such as a bag may besheared during disaggregation. In particular, the flexible container maybe configured to shear the tissue material.

Flexible containers may be used in a semi-automated or an automatedprocess for the aseptic disaggregation, stabilization and/or optionalenrichment of mammalian cells or cell aggregates.

A kit for extraction of a desired material from tissue may include adisaggregation element in which at least some tissue is treated to forma processed fluid, an enrichment element (e.g., a filter) capable ofenriching at least some of the processed fluid to form the desiredmaterial, a stabilization element capable of storing a portion of thedesired material, and an indicator tag positioned on at least one of thedisaggregation element, the enrichment element, or the stabilizationelement capable of providing at least one of a source of tissue, astatus of the tissue with respect to the process, or a identifier.

The desired material may be biological material or components of aparticular size. For example, the desired material may be tumorinfiltrating lymphocytes (TILs).

Different types of media may be used in the various processes conductedby the disaggregation element and the stabilization element. Forexample, a cryopreservation media may be provided to the kit and used inthe stabilization element to control a rate freezing.

Kit for use in a device where a disaggregation element may include afirst flexible container and the stabilization element may include asecond flexible container.

An automated device for semi-automated aseptic disaggregation and/orenrichment and/or stabilization of cells or cell aggregates frommammalian solid tissue may include a programmable processor and a kitthat includes the flexible container described herein. The automateddevice may further include an indicator tag reader. For example, anindicator tag reader may be positioned at any element (e.g.,disaggregation, enriching, or stabilization of tissue material in thekit).

In some instances, an automated device may further include radiofrequency identification tag reader to recognize samples in flexiblecontainers in the kit.

An automated device may include a programmable processor that is capableof recognizing indicators positioned on components of the kit such as abag via an indicator tag such as a QR code. After determining whichsample is in the bag, the programmable processor subsequently executes aprogram defining the type of disaggregation, enrichment, andstabilization processes and provides the respective media types requiredfor those processes.

A kit for use in an automated device may include a disaggregationflexible container or bag. The programmable processor may control adisaggregation element and disaggregation flexible container to enable aphysical and/or biological breakdown of the solid tissue.

A programmable processor may control elements of an automated devicesuch that disaggregation surfaces positioned proximate a disaggregationflexible container may mechanically crush and shear the solid tissue inthe disaggregation flexible container, optionally wherein thedisaggregation surfaces are mechanical pistons.

Disaggregation elements of a system may be controlled by a processorsuch that tissue in the disaggregation flexible container to enable aphysical and enzymatic breakdown of the solid tissue. One or more mediaenzyme solutions selected from collagenase, trypsin, lipase,hyaluronidase, deoxyribonuclease, Liberase HI, pepsin, or mixturesthereof may be provided to the disaggregation flexible container to aidin enzymatic breakdown of tissue.

A system may include a kit that includes a disaggregation flexiblecontainer and a stabilization flexible container and a programmableprocessor. The programmable processor may be adapted to control one ormore of: the disaggregation element; the enrichment element; and thestabilization element.

A programmable processor may control a stabilization element tocryopreserve the enriched disaggregated solid tissue in thestabilization container. In some embodiments, a predeterminedtemperature may be programmed.

An automated device may include additional components in a multitude ofcombinations. Components may include sensors capable of recognizingwhether a disaggregation process has been completed in thedisaggregation module prior to transfer of the disaggregated solidtissue to the optional enrichment element, weight sensors to determinean amount of media required in the containers of one or more of thedisaggregation element, an enrichment element, and/or the stabilizationelement and control the transfer of material between respectivecontainers, sensors to control temperature within the containers of theone or more of the disaggregation element; the enrichment element;and/or the stabilization element; at least one bubble sensor to controlthe transfer of media between the input and output ports of eachcontainer in the element; at least one pump, optionally a peristalticpump, to control the transfer of media between the input and outputports; pressure sensors to assess the pressure within the enrichmentelement; one or more valves to control a tangential flow filtrationprocess within the enrichment element; and/or one or more clamps tocontrol the transfer of media between the input and output ports of eachelement.

An automated device may include a programmable processor is adapted tomaintain an optimal storage temperature range in the stabilizationmodule until the container is removed. In an embodiment, theprogrammable processor may execute a controlled freezing step.

In some instances, an automated device may include a user interface. Aninterface of an automated device may include a display screen to displayinstructions that guide a user to input parameters, confirmpre-programmed steps, warn of errors, or combinations thereof.

An automated device as described herein may be adapted to betransportable.

An automatic tissue processing method may include automaticallydetermining conditions for processing steps and the associatedconditions from a digital, electronic or electromagnetic tag indicatorassociated with a component of a kit. During use a tissue sample may beplaced into a flexible container of the kit having at least one openedge. After positioning tissue in the flexible container, the open edgemay be sealed. During use tissue may be processed by automaticallyexecuting one or more tissue processing steps by communicatinginformation associated with the indicator and controlling conditionsnear the flexible container and/or positions of the flexible container.Further, addition of materials to the kit may be controlled based oninformation associated with indicators. At least some of the processedtissue may be filtered such that a filtered fluid is generated. At leastsome of the filtered fluid may be provided to a cryopreservativeflexible container to stabilize the desired material present in thefiltered fluid.

Processing as described herein may include agitation, extraction, andenzymatic digestion of at least a portion of the tissue sample in theflexible container. In some instances, this processing of tissue mayresult in the extraction of a desired material from a tissue sample. Forexample, tumor infiltrating lymphocytes (TILs) may be extracted from atissue sample.

Flexible containers, such as bags, for use in the methods describedherein may include heat-sealable material.

Tissue processing and extraction from the tissue materials using acryopreservation kit may result isolation of the desired material. Inparticular, materials such as tumor infiltrating lymphocytes (TILs) maybe the desired material.

In some instances, a cryopreservation kit and/or components thereofdescribed herein may be single use in an automated and/or asemi-automated process for the disaggregation, enrichment, and/orstabilization of cells or cell aggregates. In some embodiments, bags foruse in a cryopreservation kit such as a collection bag may in someembodiments be used for multiple processes. For example, collection bagsmay be repeatedly sealed in different locations to create separatecompartments for processing of a tissue sample such as a biopsy sampleand/or solid tissue.

Flexible containers, such as bags, for use in the invention describedherein include a collection bag and a cryopreservation bag may includeat least a portion made from a predetermined material such as athermoplastic, polyolefin polymer, ethylene vinyl acetate (EVA), blendssuch as copolymers, for example, a vinyl acetate and polyolefin polymerblend (i.e., OriGen Biomedical EVO film), a material that includes EVA,and/or coextruded layers of sealable plastics. A collection bag, such asa tissue collection bag of the invention may include a bag for receivingtissue made from a predetermined material such as ethylene vinyl acetate(EVA) and/or a material including EVA. Materials for use in the bag maybe selected for specific properties. In an embodiment, bags, includingcollection bags may be made substantially from a vinyl acetate andpolyolefin polymer blend. For example, a property of interest that maybe used to select a material for cryopreservation kit component such asa collection bag and/or the associated tubing may relate to heatsealing.

Materials for use in the bag may be selected for a specific propertyand/or a selection of properties, for example, sealability such as heatsealability, gas permeability, flexibility for example low temperatureflexibility, elasticity for example low temperature elasticity, chemicalresistance, optical clarity, biocompatibility such as cytotoxicity,hemolytic activity, resistance to leaching, having low particulates.

In some embodiments, materials may be selected for specific propertiesfor use in a coextruded material to form at least one layer of a bag.Layers may be constructed such that when constructed an interior layerof the bag is relatively biocompatible, that is the material on an innersurface of the bag is stable and does not leach into the contents of thebag.

For example, a property of interest that may be used to select amaterial for kit component such as a collection bag, a cryopreservationbag, and/or the associated tubing may relate to sealing, for exampleheat sealing.

Bags, such as collection bags and/or cryopreservation bags, and anyassociated tubing may be generally clear, transparent, translucent, anycolor desired, or a combination thereof. Tissue collection bags and/ortubing may be generally fabricated in ways analogous to the fabricationof closed and/or sealed blood and/or cryopreservation bags and theassociated tubing. Tubing in the invention may be constructed from anydesired material including, but not limited to polyvinyl chloride (PVC).For example, PVC may be a desired material as PVC is advantageous forwelding and/or sealing.

In some embodiments, at least one end of a collection bag may be openfor receiving tissue. In particular, in an embodiment, a tissue sample,for example from a biopsy may be placed in the bag through the open end,for example, a top end. In some cases, the biopsy sample may becancerous tissue from an animal (e.g., domestic animal such as dog orcat) or a human.

After tissue is positioned in the bag, the bag may be sealed, and thenmay be processed. Processing may include agitation, e.g., gentleagitation, extraction, and/or enzymatic digestion of the tissue in thebag. Tissue processing and extraction of a desired material, such astumor infiltrating lymphocytes (TILs), can be in a closed system.Advantageous or preferred embodiments may include indicators to identifythe patient from whom the tissue was collected and/or marks to showwhere the collection bag may be clamped, sealed, acted upon by a device,and/or affixed in place in an instrument.

In some embodiments, bag may be formed from a sealable material. Forexample, bag may be formed from materials including, but not limited topolymers such as synthetic polymers including aliphatic or semi-aromaticpolyamides (e.g., Nylon), ethylene-vinyl acetate (EVA) and blendsthereof, thermoplastic polyurethanes (TPU), polyethylenes (PE), a vinylacetate and polyolefin polymer blends, and/or combinations of polymers.Portions of a bag may be sealed and/or welded with energy such as heat,radio frequency energy, high frequency (HF) energy, dielectric energy,and/or any other method known in the art.

A collection bag may be used as a processing and/or disaggregation bag.Collection bags may have width in a range from about 4 cm to about 12 cmand a width in a range from about 10 cm to about 30 cm. For example, acollection bag for use in processing may have a width of about 7.8 cmand a length of about 20 cm. In particular, a bag may be heat sealable,for example, using an EVA polymer or blends thereof, a vinyl acetate andpolyolefin polymer blend, and/or one or more polyamides (Nylon).

Indicators may include, but are not limited to codes, letters, words,names, alphanumeric codes, numbers, images, bar codes, quick response(QR) codes, tags, trackers such as smart tracker tags or bluetoothtrackers, and/or any indicator known in the art. In some embodiments,indicators may be printed on, etched on, and/or adhered to a surface ofa component of a kit. Indicators may also be positioned on a hag usingan adhesive, for example, a sticker or tracker may be placed on a bagand/or on multiple bags. Collection bags and/or cryopreservation kit mayinclude multiple indicators such as numeric codes and/or QR codes.

Indicators, for example QR codes, tags such as smart tags, and/ortrackers may be used to identify a sample within a bag as well as toinstruct a device's processor such that the device runs a specificprogram according to a type of disaggregation, enrichment, and/orstabilization processes that are conducted in cryopreservation kits.Different types of media may be used in these processes, for example,enzyme media, tumor digest media and/or cryopreservation media which mayallow for a controlled rate of freezing. In some embodiments,cryopreservation kit and/or components thereof may include indicatorsthat may be readable by an automated device. The device may then executea specific fully automatic method for processing tissue when inserted tosuch a device. The invention is particularly useful in a sampleprocessing, particularly automated processing. In some instances, thecryopreservation kit and/or components thereof described herein may besingle use in an automated and/or a semi-automated process for thedisaggregation, enrichment, and/or stabilization of cells or cellaggregates. In some embodiments, bags for use in a cryopreservation kitsuch as a collection bag may in some embodiments be used for multipleprocesses. For example, collection bags may be repeatedly sealed indifferent locations to create separate compartments for processing of atissue sample such as a biopsy sample and/or solid tissue.

Further, marks may be placed at various locations on bags, such astissue collection bags to indicate where the bags may be sealed,clamped, and/or affixed to an object. In some embodiments, marks showingwhere a bag may be clamped, sealed, and/or affixed to an object, such asinstrument, may be positioned on the bag prior to use. For example, oneor more marks may be positioned on a bag during manufacturing.

Positioners may be used to ensure that tissue material in bags can betreated properly during use, for example, positioning proximate aninstrument. In some systems, the positioners may facilitate the use ofthe bags described herein in automated systems. In particular,positioners may be used to move bag through an automated system.

Use of an indicator, such as a QR code may allow for tracking of processsteps for a specific sample such that it is possible to follow thesample through a given process.

The invention involves and provides therapeutic cell populations asdiscussed in the following numbered paragraphs:

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. All rights to explicitly disclaim anyembodiments that are the subject of any granted patent(s) of applicantin the lineage of this application or in any other lineage or in anyprior filed application of any third party is explicitly reserved.Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 is a schematic diagram of a flexible container for disaggregationand digestion of the solid tissue material.

FIG. 2A is a schematic diagram of a series of filter modules that directthe digested solid tissue material to subsequent modules or a wastecontainer.

FIG. 2B is a schematic diagram of a flexible container for enrichment ofcells following digestion and removal of waste material.

FIG. 2C is a schematic diagram of another embodiment of a flexiblecontainer for enrichment of cells following digestion and removal ofwaste material.

FIG. 3A is a schematic diagram of a flexible container for stabilizationof cells following disaggregation of the solid tissue material and/orenrichment of cells.

FIG. 3B is a schematic diagram of another embodiment of a flexiblecontainer containing connections to additional flexible containers forstabilization of cells through cryopreservation following thedisaggregation of the solid tissue material and/or enrichment of cells.

FIG. 4 is a schematic diagram of the aseptic kit.

FIG. 5 is a bar graph indicating the observed fold change in apopulation of cells obtained from the disaggregation process for variousdisaggregation times ranging from a few seconds to several hours.

FIG. 6 is a diagram that describes the semi-automatic aseptic tissueprocessing method using multiple flexible containers for differentstarting solutions that are part of the modules of the process used fordisaggregation and stabilization.

FIG. 7 is a diagram that describes how flexible containers comprisingthe media used in the process may be shared between the modules of theaseptic processing kit and method.

FIG. 8 depicts a general overview of the method for the generation ofTILs.

FIG. 9 depicts an overview of the collection and processing of the tumorstarting material.

FIG. 10 depicts an overview of the TIL manufacturing process.

FIG. 11A shows a view of an embodiment of kit for processing and storingtissue materials.

FIG. 11B shows a view of an embodiment of kit for processing and storingtissue materials.

FIG. 11C shows a view of an embodiment of kit for processing and storingtissue materials.

FIG. 11D shows a view of an embodiment of kit for processing and storingtissue materials.

FIG. 12A shows a perspective view of an embodiment of a collection bag.

FIG. 12B shows a perspective view of an embodiment of a collection bag.

FIG. 12C shows a perspective view of an embodiment of a collection bag.

FIG. 12D shows a perspective view of an embodiment of a collection bag.

FIG. 12E shows a perspective view of an embodiment of a collection bag.

FIG. 13A shows a front view of an embodiment of a collection bag.

FIG. 13B shows a front view of an embodiment of a collection bag.

FIG. 13C shows a front view of an embodiment of a collection bag.

FIG. 13D shows a front view of an embodiment of a collection bag.

FIG. 13E shows a front view of an embodiment of a collection bag.

FIG. 14 shows a back view of an embodiment of a collection bag.

FIG. 15 shows a side view of an embodiment of a collection bag.

FIG. 16A shows a top view of an embodiment of a collection bag.

FIG. 16B shows a bottom view of an embodiment of a collection bag.

FIG. 17A shows a top view of an embodiment of a partially open tissuecollection bag for sealing tissue therein for processing of theinvention where the bag has sealed edges.

FIG. 17B shows a bottom view of an embodiment of an open tissuecollection bag for sealing tissue therein for processing of theinvention where the bag has sealed edges.

FIG. 18A shows a top view of an embodiment of a partially open tissuecollection bag for sealing tissue therein for processing of theinvention.

FIG. 18B shows a top view of an embodiment of a fully open tissuecollection bag for sealing tissue therein for processing of theinvention.

FIG. 19A shows a top view of an embodiment of a partially open tissuecollection bag for sealing tissue therein for processing of theinvention where the bag has sealed edges having a predetermined width.

FIG. 19B shows a top view of an embodiment of a fully open tissuecollection bag for sealing tissue therein for processing of theinvention where the bag has sealed edges having a predetermined width.

FIG. 20A shows a front view of an embodiment of a collection bag.

FIG. 20B shows a front view of an embodiment of a collection hag.

FIG. 20C shows a front view of an embodiment of a collection bag.

FIG. 20D shows a front view of an embodiment of a collection bag.

FIG. 20E shows a front view of an embodiment of a collection bag.

FIG. 21A shows a front view of an embodiment of a collection bag.

FIG. 21B shows a front view of an embodiment of a collection bag.

FIG. 21C shows a front view of an embodiment of a collection bag.

FIG. 21D shows a front view of an embodiment of a collection bag.

FIG. 21E shows a front view of an embodiment of a collection bag.

FIG. 22A shows a front view of an embodiment of a collection bag.

FIG. 22B shows a front view of an embodiment of a collection bag.

FIG. 22C shows a front view of an embodiment of a collection bag.

FIG. 22D shows a front view of an embodiment of a collection bag.

FIG. 23 shows a front view of an embodiment of a collection bag.

FIG. 24 shows a front view of an embodiment of a collection bag.

FIG. 25 shows a front view of an embodiment of a collection bag.

FIG. 26 shows a front view of an embodiment of a collection bag coupledto tubing and a port.

FIG. 27A shows a front view of an embodiment of a collection bag priorto use.

FIG. 27B shows a front view of an embodiment of a collection bag thathas been sealed, for example, after deposition of material within thehag.

FIG. 28 shows a top view of an embodiment of a cryopreservation kitfacing upwards including an open collection bag and a cryopreservationbag.

FIG. 29 shows a top view of an embodiment of a cryopreservation kitfacing downwards including a collection bag indicating where it is to beclosed and a cryopreservation bag.

FIG. 30 shows a top view of an embodiment of a cryopreservation kitfacing upwards including a closed collection bag and a cryopreservationbag.

FIG. 31 shows a side view of an embodiment of a cryopreservation kitfacing upwards including a closed collection bag and a cryopreservationbag.

FIG. 32 shows an end view of an embodiment of a cryopreservation kit.

FIG. 33 shows a top view of an embodiment of a collection hag includingindicia coupled to tubing.

FIG. 34 shows a front view of an embodiment of a cryopreservation kitthat includes a collection bag, a filter, and a cryopreservation bag.

FIG. 35 shows a front view of an embodiment of a cryopreservation kitthat includes a collection bag, a filter, and a cryopreservation bag.

FIG. 36A shows a front view of an embodiment of a cryopreservation kitthat includes a collection bag, a filter, and a cryopreservation bag.

FIG. 36B shows a side view of an embodiment of a collection bag securedusing a clamp, hinge, and latch as well as a bar positioned to proximatea surface of the collection bag during use.

FIG. 36C shows an exploded view of a clamp positioned on a collectionbag.

FIG. 37 shows a front view of an embodiment of a cryopreservation kitthat includes a collection bag, a filter, and a cryopreservation bag.

FIG. 38 shows a front view of an embodiment of a cryopreservation kitthat includes a collection bag, a filter, and a cryopreservation bag.

FIG. 39 shows a front view of an embodiment of a collection bag securedby a clamp.

FIG. 40 shows a front view of an embodiment of a collection bag.

FIG. 41 shows a front view of a treading device for the disaggregationof tissue into individual cells or cell clumps within a closed samplecontainer.

FIG. 42 and FIG. 43 show the device of FIG. 41 in two differentrespective operational positions;

FIG. 44 shows a plan view of the device shown in the previous Figures.

FIG. 45 shows another plan view of an alternative construction of thedevice.

FIG. 46 , FIG. 47 and FIG. 48 show three different constructions of asample container suitable for use with the device of FIGS. 41 to 45 ,

FIG. 49 shows a sample bag being prepared for use.

FIG. 50 , FIG. 51A, FIG. 51B, and FIG. 51C show alternative ways ofsealing the sample bag.

FIG. 52 , FIG. 53 , and FIG. 54 show apparatus and techniques forpreparing the bag for use.

FIG. 55 shows loading of the sample bag or container into the treadingdevice.

FIG. 56 , FIG. 57 , and FIG. 58 show apparatus for dividing adisaggregated sample.

FIG. 59 , FIG. 60 , and FIG. 61 show apparatus for controlling thetemperature of a sample or divided sample.

FIG. 62 , FIG. 63 , and FIG. 64 show a further embodiment of a treadingdevice.

FIG. 65 is an exemplary flow diagram for collection, processing andcryopreservation of tumor tissue.

FIG. 66 is an exemplary flow diagram for TIL manufacture from processedand cryopreserved tumor tissue.

FIG. 67A-67C compare yield (FIG. 67A), percent viability (FIG. 67B), andpercent CD3+ T cells (FIG. 67C) of cryopreserved and fresh disaggregatedcell suspensions.

FIGS. 68A and 68B compare viability of PBMCs cryopreserved withcommercially available cryopreservants.

FIG. 69 compares viability of PBMCs digested then cryopreservedfollowing a protocol that held the material at 4° C. for 10 minutes,then decreased the temperature at a rate of −1° C./min or decreased from35° C. to −80° C. directly at a rate of −2° C./min.

FIG. 70 compares temperatures recorded from sample bags following aprotocol that held the material at 4° C. for 10 minutes, then decreasedthe temperature at a rate of −1° C./min or decreased from 35° C. to −80°C. directly at a rate of −2° C./min.

FIG. 71A-71D and FIG. 71E-71H depict disaggregation and cryopreservationof TIL077: FIG. 71A Disaggregator speed setpoint; FIG. 71B Disaggregatorspeed record; FIG. 71C Temperature setpoint (disaggregation); FIG. 71DCryo-plate temperature record (disaggregation);

FIG. 71E Temperature setpoint (cryopreservation); FIG. 71F Temperaturerecord (cryopreservation); FIG. 71G Setpoint cooling rate; FIG. 71HCryo-plate cooling rate record.

FIG. 72A-72D and FIG. 72E-72H depict Tiss-U-Stor disaggregation andcryopreservation of TIL078 (1 of 2 bags): FIG. 72A Disaggregator speedsetpoint; FIG. 72B Disaggregator speed record; FIG. 72C Temperaturesetpoint (disaggregation); FIG. 72D Cryo-plate temperature record(disaggregation); FIG. 72E Temperature setpoint (cryopreservation);

FIG. 72F Temperature record (cryopreservation); FIG. 72G Setpointcooling rate; FIG. 72H Cryo-plate cooling rate record.

FIG. 73A-73C and FIG. 73D-73F depict Tiss-U-Stor disaggregation andcryopreservation of TIL078 in a continuous process: FIG. 73ADisaggregator speed setpoint; FIG. 73B Disaggregator speed record; FIG.73C Temperature setpoint (disaggregation and cryopreservation); FIG. 73DCryo-plate temperature record (disaggregation and cryopreservation);FIG. 73E Cooling rate setpoint (disaggregation and (cryopreservation);FIG. 73F Cryo-plate cooling rate record (disaggregation and(cryopreservation).

FIG. 74 depicts a waterfall plot showing best overall response andpercent change in tumor burden. CR, complete response; PD, progressivedisease; PR, partial response; SD, stable disease. The tumor burden isdefined as the sum of the diameters of the target lesions; The change intumor burden is defined as the change from baseline to post-baselinenadir. A minimum post-baseline SLD of 0 was used in both CR patients,who did not have target lesion measures reported at the visits when CRwas assessed (no disease or metastasis was observed through CT/MRIscans). One subject with a best overall response of PD did not have anypost-treatment target lesion measures reported (progression determinedby observation of new lesions) and hence was not presented in the plot.

FIG. 75A-75C depict overall survival time. FIG. 75A The median overallsurvival (OS) time with all 21 treated patients was 21.3 months. FIG.75B The median OS time of 15 patients with quantitative response datawas 16 months. FIG. 75C The median OS time for nonresponders (N=7) was6.5 months. The median OS time for responders (per quantitative responseonly, N=8) was not reached.

FIG. 76A, FIG. 76B and FIG. 76C depict characteristics of manufacturedTILs. (A) Cell count during TIL outgrowth stage (stage 1) of thefull-scale ITIL-168 GMP runs. (B) Cell count during TIL REP stage (stage2) of the full-scale ITIL-168 GMP runs. (C) Percent viability (% viableCD3+ cells) during the full-scale ITIL-168 GMP runs.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs.

The term “anti-CD3 antibody” refers to an antibody or variant thereof,e.g., a monoclonal antibody and including human, humanized, chimeric,murine or mammalian antibodies which are directed against the CD3receptor in the T cell antigen receptor of mature human T cells.Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3antibodies also include the UHCT1 clone, also known as T3 andCD3.epsilon. Other anti-CD3 antibodies include, for example,otelixizumab, teplizumab, and visilizumab.

When “an anti-tumor effective amount”, “an tumor-inhibiting effectiveamount”, or “therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). It can generally be stated that apharmaceutical composition comprising the tumor infiltrating lymphocytes(e.g. secondary TILs or genetically modified cytotoxic lymphocytes)described herein may be administered at a dosage of 10⁴ to 10¹¹ cells/kgbody weight (e.g., 10⁵ to 10⁶, 10 ⁵ to 10¹⁰, 10 ⁵ to 10¹¹, 10 ⁶ to 10¹⁰,10 ⁶ to 10¹¹, 10 ⁷ to 10¹¹, 10 ⁷ to 10¹⁰, 10 ⁸ to 10¹¹, 10 ⁸ to 10¹⁰, 10⁹ to 10¹¹, or 10⁹ to 10¹⁰ cells/kg body weight), including all integervalues within those ranges. Tumor infiltrating lymphocytes (including insome cases, genetically modified cytotoxic lymphocytes) compositions mayalso be administered multiple times at these dosages. The tumorinfiltrating lymphocytes (including in some cases, genetically) can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particularpatient can readily be determined by one skilled in the art of medicineby monitoring the patient for signs of disease and adjusting thetreatment accordingly.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients (in a preferred embodiment of thepresent invention, for example, at least one potassium channel agonistin combination with a plurality of TILs) to a subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the subject at the same time. Co-administration includes simultaneousadministration in separate compositions, administration at differenttimes in separate compositions, or administration in a composition inwhich two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administrationin a composition in which both agents are present are preferred.

“Cellularized or cellularization” as used herein refers to the processof disaggregation where by the solid tissue a multicellular materialgenerally made up of multiple cell lineages/types is broken down intosmall numbers of cells including but not limited to one cell but couldbe multiple cells of various lineages or cell types in very smallnumbers i.e. clump of cells or cell aggregates.

“Closed system” as used herein refers to a system that is closed to theoutside environment. Any closed system appropriate for cell culturemethods can be employed with the methods of the present invention.Closed systems include, for example, but are not limited to closed G-Rexcontainers or cell culture bags. Once a tumor segment is added to theclosed system, the system is not open to the outside environment untilthe TILs are ready to be administered to the patient. In an advantageousembodiment, the closed system is the system disclosed in PCT PublicationNo. WO 2018/130845.

“Cryopreservation media” or “cryopreservation medium” as used hereinrefers to any medium that can be used for cryopreservation of cells.Such media can include media comprising 2% to 10% DMSO. Exemplary mediainclude CryoStor CS10, HypoThermosol, Bloodstor BS-55 as well ascombinations thereof.

The term “Cryopreserved TILs” herein is meant that TILs, either primary,bulk, or expanded (REP TILs), are treated and stored in the range ofabout −190° C. to −60° C. General methods for cryopreservation are alsodescribed elsewhere herein, including in the Examples. For clarity,“cryopreserved TILs” are distinguishable from frozen tissue sampleswhich may be used as a source of primary TILs.

“Depletion” as used herein refers to a process of a negative selectionthat separates the desired cells from the undesired cells which arelabelled by one marker-binding fragment coupled to a solid phase.

“Disaggregation or disaggregate” as used herein refers to thetransformation of solid tissue into a single cells or small cell numberaggregates where a single cell as a spheroid has a diameter in the rangeof 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60μm, 70 μm, 80 μm, 90 μm, 100 μm, or more, wherein this is more usuallybetween 7 to 20 μm.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated (e.g., theweight, age and gender of the subject), the severity of the diseasecondition, or the manner of administration. The term also applies to adose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

“Engineered” as used herein refers to either addition of nucleicmaterial or factors, which change the tissue derived cell function fromtheir original function to have a new or improved function for itsultimate utility.

“Enzyme Media” as used herein refers to media having enzymatic activitysuch as collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease,Liberase HI, pepsin, or mixtures thereof.

“Filtrate” as used herein refers to the material that passes through afilter, mesh or membrane.

“Flexible container” as used herein refers to a flexible packagingsystem in multiple formats with one or more different types of film.Each film type is selected to provide specific characteristics topreserve the physical, chemical, and functional characteristics of thesterile fluids, solid tissue derived cellular material and the containerintegrity depending upon the step of the process.

“Freezing solution” or “cryopreservation solution” also referred in thefield to as the cryoprotectant is a solution that containscryoprotective additives. These are generally permeable, non-toxiccompounds which modify the physical stresses cells are exposed to duringfreezing in order to minimize freeze damage (i.e. due to ice formation)and are most commonly a % vol/vol of one or more of the following:dimethylsulphoxide (DMSO); ethylene glycol; glycerol;2-methyl-2,4-pentanediol (MPD); propylene glycol; sucrose; andtrehalose.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CIVIL), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

The term “IL-2” (also referred to herein as “IL2”) refers to the T cellgrowth factor known as interleukin-2, and includes all forms of IL-2including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-2 isdescribed, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek,Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which areincorporated by reference herein. The amino acid sequence of recombinanthuman IL-2 suitable for use in the invention is given in Table 2 (SEQ IDNO: 3). For example, the term IL-2 encompasses human, recombinant formsof IL-2 such as aldesleukin (PROLEUKIN, available commercially frommultiple suppliers in 22 million IU per single use vials), as well asthe form of recombinant IL-2 commercially supplied by CellGenix, Inc.,Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd.,East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercialequivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125human IL-2) is a nonglycosylated human recombinant form of IL-2 with amolecular weight of approximately 15 kDa. The term IL-2 also encompassespegylated forms of 1L-2, as described herein, including the pegylatedIL2 prodrug NKTR-214, available from Nektar Therapeutics, South SanFrancisco, Calif, USA. NKTR-214 and pegylated IL-2 suitable for use inthe invention is described in U.S. Patent Application Publication No. US2014/0328791 A1 and International Patent Application Publication No. WO2012/065086 A1. Alternative forms of conjugated IL-2 suitable for use inthe invention are described in U.S. Pat. Nos. 4,766,106, 5,206,344,5,089,261 and 4,902,502. Formulations of IL-2 suitable for use in theinvention are described in U.S. Pat. No. 6,706,289.

The term “IL-4” (also referred to herein as “IL4”) refers to thecytokine known as interleukin 4, which is produced by Th2 T cells and byeosinophils, basophils, and mast cells. IL-4 regulates thedifferentiation of naive helper T cells (Th0 cells) to Th2 T cells.Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation byIL-4, Th2 T cells subsequently produce additional IL-4 in a positivefeedback loop. IL-4 also stimulates B cell proliferation and class IIMHC expression, and induces class switching to IgE and IgG1 expressionfrom B cells. Recombinant human IL-4 suitable for use in the inventionis commercially available from multiple suppliers, includingProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No.CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (humanIL-15 recombinant protein, Cat. No. Gibco CTP0043).

The term “IL-7” (also referred to herein as “IL7”) refers to aglycosylated tissue-derived cytokine known as interleukin 7, which maybe obtained from stromal and epithelial cells, as well as from dendriticcells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate thedevelopment of T cells. IL-7 binds to the IL-7 receptor, a heterodimerconsisting of TL-7 receptor alpha and common gamma chain receptor, whichin a series of signals important for T cell development within thethymus and survival within the periphery. Recombinant human 1L-7suitable for use in the invention is commercially available frommultiple suppliers, including ProSpec-Tany TechnoGene Ltd., EastBrunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific,Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No.Gibco PHC0071).

The term “IL-12” (also referred to herein as “IL12”) refers to the Tcell growth factor known as interleukin-12. Interleukin (IL)-12 is asecreted heterodimeric cytokine comprised of 2 disulfide-linkedglycosylated protein subunits, designated p35 and p40 for theirapproximate molecular weights. IL-12 is produced primarily byantigen-presenting cells and drives cell-mediated immunity by binding toa two-chain receptor complex that is expressed on the surface of T cellsor natural killer (NK) cells. The IL-12 receptor beta-1 (IL-12Rpi) chainbinds to the p40 subunit of IL-12, providing the primary interactionbetween IL-12 and its receptor. However, it is IL-12p35 ligation of thesecond receptor chain, IL-12RP2, that confers intracellular signaling.IL-12 signaling concurrent with antigen presentation is thought toinvoke T cell differentiation towards the T helper 1 (Thl) phenotype,characterized by interferon gamma (IFNy) production. Thl cells arebelieved to promote immunity to some intracellular pathogens, generatecomplement-fixing antibody isotypes, and contribute to tumorimmunosurveillance. Thus, IL-12 is thought to be a significant componentto host defense immune mechanisms. IL-12 is part of the IL-12 family ofcytokines which also includes IL-23, TL-27, IL-35, IL-39.

The term “IL-15” (also referred to herein as “IL15”) refers to the Tcell growth factor known as interleukin-15, and includes all forms ofIL-15 including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-15 isdescribed, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, thedisclosure of which is incorporated by reference herein. IL-15 shares 13and y signaling receptor subunits with IL-2. Recombinant human IL-15 isa single, non-glycosylated polypeptide chain containing 114 amino acids(and an N-terminal methionine) with a molecular mass of 12.8 kDa.Recombinant human IL-15 is commercially available from multiplesuppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J.,USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham,Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82).

The term “IL-18” (also referred to herein as “IL18”) refers to the Tcell growth factor known as interleukin-15. Interleukin-18 (IL-18) is aproinflammatory cytokine that belongs to the 1L-1 cytokine family, dueto its structure, receptor family and signal transduction pathways.Related cytokines include IL-36, IL-37, IL-38.

The term “IL-21” (also referred to herein as “IL21”) refers to thepleiotropic cytokine protein known as interleukin-21, and includes allforms of IL-21 including human and mammalian forms, conservative aminoacid substitutions, glycoforms, biosimilars, and variants thereof. IL-21is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the disclosure of which is incorporated by reference herein.IL-21 is primarily produced by natural killer T cells and activatedhuman CD4+ T cells. Recombinant human IL-21 is a single,non-glycosylated polypeptide chain containing 132 amino acids with amolecular mass of 15.4 kDa. Recombinant human 1L-21 is commerciallyavailable from multiple suppliers, including ProSpec-Tany TechnoGeneLtd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein,Cat. No. 14-8219-80).

The term “liquid tumor” refers to an abnormal mass of cells that isfluid in nature. Liquid tumor cancers include, but are not limited to,leukemias, myelomas, and lymphomas, as well as other hematologicalmalignancies. TILs obtained from liquid tumors may also be referred toherein as marrow infiltrating lymphocytes (MILs).

“Magnetic” in “magnetic particle” as used herein refers to all subtypesof magnetic particles, which can be prepared with methods well known tothe skilled person in the art, especially ferromagnetic particles,superparamagnetic particles and paramagnetic particles. “Ferromagnetic”materials are strongly susceptible to magnetic fields and are capable ofretaining magnetic properties when the field is removed. “Paramagnetic”materials have only a weak magnetic susceptibility and when the field isremoved quickly lose their weak magnetism. “Superparamagnetic” materialsare highly magnetically susceptible, i.e. they become strongly magneticwhen placed in a magnetic field, but, like paramagnetic materials,rapidly lose their magnetism.

“Marker” as used herein refers to a cell antigen that is specificallyexpressed by a certain cell type. Preferentially, the marker is a cellsurface marker, so that enrichment, isolation and/or detection of livingcells can be performed.

“Marker-binding fragment” as used herein refers to any moiety that bindspreferentially to the desired target molecule of the cell, i.e. theantigen. The term moiety comprises, e.g., an antibody or antibodyfragment. The term “antibody” as used herein refers to polyclonal ormonoclonal antibodies which can be generated by methods well known tothe person skilled in the art. The antibody may be of any species, e.g.murine, rat, sheep, human. For therapeutic purposes, if non-humanantigen binding fragments are to be used, these can be humanized by anymethod known in the art. The antibodies may also be modified antibodies(e.g. oligomers, reduced, oxidized and labelled antibodies). The term“antibody” comprises both intact molecules and antibody fragments, suchas Fab, Fab′, F(ab′)2, Fv and single-chain antibodies. Additionally, theterm “marker-binding fragment” includes any moiety other than antibodiesor antibody fragments that binds preferentially to the desired targetmolecule of the cell. Suitable moieties include, without limitation,oligonucleotides known as aptamers that bind to desired target molecules(Hermann and Pantel, 2000: Science 289: 820-825), carbohydrates, lectinsor any other antigen binding protein (e.g. receptor-ligand interaction).

“Media” means various solutions known in the art of cell culturing, cellhandling and stabilization used to reduce cell death, including but notlimited to one or more of the following media Organ PreservationSolutions, selective lysis solutions, PBS, DMEM, HBSS, DPBS, RPMI,Iscove's medium, X-VIVO™, Lactated Ringer's solution, Ringer's acetate,saline, PLASMALYTE™ solution, crystalloid solutions and IV fluids,colloid solutions and IV fluids, five percent dextrose in water (D5W),Hartmann's Solution. The media can be standard cell media like the abovementioned-media or special media for e.g. primary human cell culture(e.g. for endothelia cells, hepatocytes, or keratinocytes) or stem cells(e.g. dendritic cell maturation, hematopoietic expansion, keratinocytes,mesenchymal stem cells or T cell expansion). The media may havesupplements or reagents well known in the art, e.g. albumins andtransport proteins, amino acids and vitamins, antibiotics, attachmentsfactors, growth factors and cytokines, hormones, metabolic inhibitors orsolubilizing agents. Various media are commercially available e. g. fromThermoFisher Scientific or Sigma-Aldrich.

The term “microenvironment,” as used herein, may refer to the solid orhematological tumor microenvironment as a whole or to an individualsubset of cells within the microenvironment. The tumor microenvironment,as used herein, refers to a complex mixture of “cells, soluble factors,signaling molecules, extracellular matrices, and mechanical cues thatpromote neoplastic transformation, support tumor growth and invasion,protect the tumor from host immunity, foster therapeutic resistance, andprovide niches for dominant metastases to thrive,” as described inSwartz, et al., Cancer Res., 2012, 72, 2473. Although tumors expressantigens that should be recognized by T cells, tumor clearance by theimmune system is rare because of immune suppression by themicroenvironment.

The term “negatively separated” as used herein refers to the activeseparation of cells which are bound by one marker-binding fragmentcoupled to a solid phase and these cells are not the required populationof cells.

“Non-labelled” or “untouched” as used herein refers to the cells whichare not bound by one marker-binding fragment coupled to a solid phase.The non-labelled, untouched cell fraction contains the desired targetcells.

“Non-target cells” as used herein refers to cells which are specificallybound by one marker-binding fragment which is coupled to a solid phasethat is used to remove an unwanted cell type.

“OKT-3” (also referred to herein as “OKT3”) refers to a monoclonalantibody or biosimilar or variant thereof, including human, humanized,chimeric, or murine antibodies, directed against the CD3 receptor in theT cell antigen receptor of mature T cells, and includescommercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure,Miltenyi Biotech, Inc., San Diego, Calif, USA) and muromonab orvariants, conservative amino acid substitutions, glycoforms, orbiosimilars thereof.

“Particle” as used herein refers to a solid phase such as colloidalparticles, microspheres, nanoparticles, or heads. Methods for generationof such particles are well known in the field of the art. The particlesmay be magnetic particles or have other selective properties. Theparticles may be in a solution or suspension or they may be in alyophilized state prior to use in the present invention. The lyophilizedparticle is then reconstituted in convenient buffer before contactingthe sample to be processed regarding the present invention.

The terms “peripheral blood mononuclear cells” and “PBMCs” refers to aperipheral blood cell having a round nucleus, including lymphocytes (Tcells, B cells, NK cells) and monocytes. Preferably, the peripheralblood mononuclear cells are irradiated allogeneic peripheral bloodmononuclear cells. PBMCs are a type of antigen-presenting cell.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” are intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and inert ingredients. The useof such pharmaceutically acceptable carriers or pharmaceuticallyacceptable excipients for active pharmaceutical ingredients is wellknown in the art. Except insofar as any conventional pharmaceuticallyacceptable carrier or pharmaceutically acceptable excipient isincompatible with the active pharmaceutical ingredient, its use in thetherapeutic compositions of the invention is contemplated. Additionalactive pharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

The term “population of cells” (including TILs) herein is meant a numberof cells that share common traits. In general, populations generallyrange from 1×10⁶ to 1×10¹² in number, with different TIL populationscomprising different numbers.

“Positively separated” as used herein refers to the active separation ofcells which are bound by one marker-binding fragment coupled to a solidphase and these cells are the required population of cells.

“Negatively separated” as used herein refers to the active separation ofcells which are bound by one marker-binding fragment coupled to a solidphase and these cells are not the required population of cells.

“Purity” as used herein refers to the percentage of the targetpopulation or populations desired from the original solid tissue.

“Rapid expansion” means an increase in the number of antigen-specificTILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over aperiod of a week, more preferably at least about (or 20-, 30-, 40-, 50-,60-, 70-, 800-, or 90-fold) over a period of a week, more preferably atleast about 100-fold (or 200-, 300-, 400-, 500-, 600-, 700-, 800-, or900-fold) over a period of a week, or most preferably at least about1000-fold or 2000-, 3000-, 4000-, 5000-, 6000-, 7000-, 8000-, or9000-fold) over a period of a week. A number of rapid expansionprotocols are outlined below.

“Regenerative medicine(s)”, “adoptive cell therapy(ies)” or “advancedtherapy medicinal product(s)” are used interchangeably herein to referto cellular material that is used for therapeutic purposes of one ormore mammals either by: the action of a part of or all of the cellularmaterial; the supportive actions of a part of or all of the cellularmaterial with the aim to improve the wellbeing of the mammal afterapplication. The therapeutic cells can either be used directly or mayrequire further processing, expansion and/or engineering to providethese actions.

“Sample” as used herein refers to a sample containing cells in anyratio. Preferentially, these cells are viable. In some instances, thesecells can also be fixed or frozen cells which may be used for subsequentnucleic acids or protein extraction. The samples may be from animals,especially mammals such as mouse, rats, or humans. Any compressiblesolid tissue that contains cells can be used. The invention isillustrated mainly through the isolation of hematopoietic and cancercells from solid tumor tissue. However, the invention relates to amethod for isolation of a breadth of cells from any mammalian solidtissue.

“Solid phase” as used herein refers to the coupling of themarker-binding fragment, e.g. an antibody, bound to anothersubstrate(s), e.g. particles, fluorophores, haptens like biotin,polymers, or larger surfaces such as culture dishes and microtiterplates. In some cases, the coupling results in direct immobilization ofthe antigen-binding fragment, e.g. if the antigen-binding fragment iscoupled to a larger surface of a culture dish. In other cases, thiscoupling results in indirect immobilization, e.g. an antigen-bindingfragment coupled directly or indirectly (via e.g. biotin) to a magneticbead is immobilized if said bead is retained in a magnetic field. Infurther cases the coupling of the antigen-binding fragment to othermolecules results not in a direct or indirect immobilization but allowsfor enrichment, separation, isolation, and detection of cells accordingto the present invention, e.g. if the marker-binding fragment is coupledto a chemical or physical moiety which then allows discrimination oflabelled cells and non-labelled cells, e.g. via flow cytometry methods,like FACS sorting, or fluorescence microscopy.

“Solid tissue” as used herein refers to a piece or pieces of animalderived mammalian solid tissue which by its three dimensions i.e.length, breadth and thickness as a geometrical body is larger than thesize of multiple individual cell based units and often containsconnective materials such as collagen or a similar matrix that make upstructure of the tissue whereby said solid tissue cannot flow throughtubes or be collected by a syringe or similar small conduit orreceptacle and is i.e. with dimensions in the range of 500 μm, 1 mm, 2mm, 3 mm, 4 mm, 5 mm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 10 cm, 20 cm, 30 cm,or more.

“Solid tumor” refers to an abnormal mass of tissue that usually does notcontain cysts or liquid areas. Solid tumors may be benign or malignant.The term “solid tumor cancer” refers to malignant, neoplastic, orcancerous solid tumors. Solid tumor cancers include, but are not limitedto, sarcomas, carcinomas, and lymphomas, such as cancers of the lung,breast, prostate, colon, rectum, and bladder. The tissue structure ofsolid tumors includes interdependent tissue compartments including theparenchyma (cancer cells) and the supporting stromal cells in which thecancer cells are dispersed and which may provide a supportingmicroenvironment. In some embodiments, the cancer is selected fromcervical cancer, head and neck cancer (including, for example, head andneck squamous cell carcinoma [HNSCC]) glioblastoma, ovarian cancer,sarcoma, pancreatic cancer, bladder cancer, breast cancer, triplenegative breast cancer, and non-small cell lung carcinoma. The tissuestructure of solid tumors includes interdependent tissue compartmentsincluding the parenchyma (cancer cells) and the supporting stromal cellsin which the cancer cells are dispersed and which may provide asupporting microenvironment.

By “thawed cryopreserved TILs” herein is meant a population of TILs thatwas previously cryopreserved and then treated to return to roomtemperature or higher, including but not limited to cell culturetemperatures or temperatures wherein TILs may be administered to apatient.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its developmentor progression; and (c) relieving the disease, i.e., causing regressionof the disease and/or relieving one or more disease symptoms.“Treatment” is also meant to encompass delivery of an agent in order toprovide for a pharmacologic effect, even in the absence of a disease orcondition. For example, “treatment” encompasses delivery of acomposition that can elicit an immune response or confer immunity in theabsence of a disease condition, e.g., in the case of a vaccine.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8+ cytotoxic T cells (lymphocytes),Thi and Thi 7 CD4+ T cells, natural killer cells, dendritic cells, andMl macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to hulkTTLs and expanded TTLs (“REP TTLs” or “post-REP TILs”). TIL cellpopulations can include genetically modified TILs. TILs can generally bedefined either biochemically, using cell surface markers, orfunctionally, by their ability to infiltrate tumors and effecttreatment. TILs can be generally categorized by expressing one or moreof the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7,CD45Ra, CD62L, CD95, PD-1, and CD25. Additionally and alternatively,TILs can be functionally defined by their ability to infiltrate solidtumors upon reintroduction into a patient. TILS may further becharacterized by potency—for example, TILS may be considered potent orfunctional if in response to TCR engagement they produce, for example,interferon (IFN) release is greater than about 50 pg/mL, greater thanabout 100 pg/mL, greater than about 150 pg/mL, or greater than about 200pg/mL, or more preferably individual cells can be Potency throughintracellular staining for CD137, CD107a, INF-y TNF-a, and IL-2following TCR induced stimulation by flow cytometry.

“Retentate” as used herein refers to the material that does not passthrough a filter, mesh or membrane.

“Ultimate utility” as used herein refers to manufacture of or direct usein regenerative medicines, adoptive cell therapies, ATMPs, diagnostic invitro studies or scientific research.

The present invention relates to tumor infiltrating lymphocytes (TILs)in particularly unmodified TILS (UTILs), which may be isolated fromtumors of a metastatic cancer patient, involving autologous TILsgenerated from and returned to the same cancer patient. The presentinvention also relates to methods for isolating a therapeutic populationof cryopreserved TILs or UTILs and to TILs and UTILs obtained orobtainable via use of a device comprising a single use aseptic kit forprocessing of a resected tumor by the methods described herein.

In general, TILs are initially obtained from a patient tumor sample(“primary TILs”) and then expanded into a larger population for furthermanipulation as described herein, cryopreserved, restimulated asoutlined herein and optionally evaluated for phenotype and metabolicparameters as an indication of TIL health.

A patient tumor sample may be obtained using methods known in the art,generally via surgical resection, needle biopsy or other means forobtaining a sample that contains a mixture of tumor and TIL cells. Ingeneral, the tumor sample may be from any solid tumor, including primarytumors, invasive tumors or metastatic tumors. The tumor sample may alsobe a liquid tumor, such as a tumor obtained from a hematologicalmalignancy. The solid tumor may be of any cancer type, including, butnot limited to, breast, ovary, cervical, pancreatic, prostate,colorectal, lung, brain, renal, stomach, and skin (including but notlimited to squamous cell carcinoma, basal cell carcinoma, and melanoma).In some embodiments, TILs are obtained from malignant melanoma tumors,as these have been reported to have particularly high levels of TILs.

The production generally involves a two-stage process. In stage 1,initial tumor material is dissected, placed in the aseptic kit having adisaggregation module, enzymatically digesting and/or fragmenting, andhomogenizing the tumor in the disaggregation module to provide a singlecell suspension. While the homogenized cells can be further purifiedwithin the aseptic kit in a separate enrichment module to removecomponents such as no longer required reagents; cell debris;non-disaggregated tissue, the cells can be directly cryopreserved tostabilize the starting material for TIL manufacture and storage in thestabilization module of the aseptic kit until Stage 2 is required. Stage2 generally involves growth of the TILs out of the resected tumorstarting material (2 weeks), followed by a rapid expansion process ofthe TIL cells (rapid expansion protocol “REP”—2 weeks). The finalproduct is washed and harvested prior to suspension in buffered saline,8.5% HAS and 10% DMSO and cryopreserved to form a solid aseptic productthat is thawed prior to infusion as a single dose with no furthermodification.

There are three separate elements to the treatment that potentiallycontribute to therapeutic activity. The core element is the TILs i.e.tumor-derived T cells, which can target and eliminate tumor cells by avariety of methods utilized by T cells as a part of their normalfunction. These methods include direct methods (i.e. perforin-mediatedcytotoxicity) and indirect methods (i.e. cytokine production). Which ofthese methods is the most important to in vivo anti-tumor effects isunclear although mouse models suggest that the production of interferongamma is critical for effective therapy. The two other elements whichcontribute to the therapy are pre-conditioning chemotherapy and highdose intravenous IL-2. These two elements are thought to act bysupporting engraftment of T cells in the patient after infusion:initially through conditioning chemotherapy which removes competing andregulating immune cells; followed by the IL-2 component which supportssurvival of T cells.

The structure of the cell therapy product is created by growing the TILdirectly out of an enzyme digested tumor mass by means of growthsupporting cell culture media and a T cell supporting growth factorInterleukin-2 (IL-2). This enables tumor specific T cells to selectivelysurvive and grow out of the tumor cell mixture, while T cells that donot recognize tumor antigens will not be stimulated and be selectivelylost. The product comprises an autologous T-cell based product where theT cells have been derived from a patient's own cancer tissue and rapidlyexpanded to form a pure T cell population and T cells as defined by CD3surface marker.

In brief, TILs, in particular UTILs, may be produced in a two-stageprocess using a tumor biopsy as the starting material: Stage 1(generally performed over 2-3 hours) initial collection and processingof tumor material using dissection, enzymatic digestion andhomogenization via use of a kit and a semi-automatic device to produce asingle cell suspension which can be directly cryopreserved using thestabilization module of the kit to stabilize the starting material forsubsequent manufacture and Stage 2 which can occur days or years later.Stage 2 may be performed over 4 weeks, which may be a continuous processstarting with thawing of the product of Stage 1 and growth of the TILout of the tumor starting material (about 2 weeks) followed by a rapidexpansion process of the TIL cells (about 2 weeks) to increase theamount of cells and therefore dose. The TILs, in particular UTILs, areconcentrated and washed prior to formulation as a liquid suspension ofcells. The aseptic drug product may be cryopreserved in a bag that willbe thawed prior to intravenous infusion as a single dose with no furthermodification.

In one embodiment, a bag of the invention is a collection bag and/or acryopreservation bag. Bags and any associated tubing may be generallyclear, transparent, translucent, any color desired, or a combinationthereof. Tissue collection bags and/or tubing may be generallyfabricated in ways analogous to the fabrication of closed and/or sealedblood and/or cryopreservation bags and the associated tubing. Tubing inthe invention may be constructed from any desired material including,but not limited to polyvinyl chloride (PVC). For example, PVC may be adesired material as PVC is advantageous for welding and/or sealing.

A collection bag, such as a tissue collection bag of the invention mayinclude at least a portion of the bag for receiving tissue made from apredetermined material such as a polyolefin polymer, ethylene vinylacetate (EVA), copolymers such as vinyl acetate and polyolefin polymerblend (i.e., OriGen Biomedical EVO film), and/or a material includingEVA. Materials for use in the bag may be selected for a specificproperty and/or a selection of properties, for example, salability suchas heat sealability, gas permeability, flexibility for example lowtemperature flexibility, elasticity for example low temperatureelasticity, chemical resistance, optical clarity, biocompatibility suchas cytotoxicity, hemolytic activity, resistance to leaching, having lowparticulate.

Seals may be formed during use with energy, for example, heat to createa weld zone. Seals formed during use may be have a width in a range fromabout 2.5 mm to about 7.5 mm. Generally, seal 140 is formed after tissuematerial is placed in bag 140 and may have a width of about 5 mm. Sealsmay be tested for strength using a seal peel test (i.e., ASTM F88/F88M),and/or a burst test (i.e., ASTM F1140/F1140M or ASTM F2051/F2054M).

In some embodiments, a bag or a flexible container may withstand a forceof 100 Newtons during use when properly sealed and further secured witha clamp when positioned within a device for treatment and/or processing.A bag or a flexible container embodiment may be constructed to withstanda force of 75 Newtons during use when properly sealed and furthersecured with a clamp when positioned within a device for treatmentand/or processing.

When forming seals or welds on a flexible container such as a bag, forexample, a collection bag and/or a cryopreservation bag, a sealingdevice may be used to apply heat and/or pressure at a predeterminedtemperature, pressure, and amount of time depending on the material usedin the bag. For example, some heat sealers may require application ofheat and pressure for about eight seconds. After 8 seconds, heat may beturned off on the device, however, pressure may be applied for anadditional 2 to 3 seconds.

In some embodiments, bags may have a length in a range from about 10 cmto about 50 cm. In particular, bags for use in the invention describedherein may have a length in a range from about 15 cm to about 30 cm. Forexample, bags may have a length in a range from about 18 cm to about 22cm.

Some of the tubing may be weldable. Weldable tubing may be made from apolymer material, for example, polyvinyl chloride (PVC).

Valves including, but not limited to needle free valves may be used atpoints along the tubing. In some embodiments, bags may have a length ina range from about 10 cm to about 40 cm. In particular, bags for use inthe invention described herein may have a length in a range from about15 cm to about 30 cm. For example, bags may have a length in a rangefrom about 18 cm to about 22 cm.

Cryopreservation bags may need to be suitable for cryopreservation witha cryoprotectant such as dimethyl sulfoxide (“DMSO”). In someembodiments, cryopreservation bags may be constructed so that the bagsmay hold a volume of material in a range from about 5 ml to about 45 ml.In particular, a cryopreservation bag may include accommodate a volumeof material in a range from about 10 ml to about 35 ml. For example,some embodiments include cryopreservation bags that may accommodate avolume of material to be stored in a range from about 15 ml to about 30ml. A cryopreservation bag may have sized such that a desiredpredetermined volume is achieved. In some embodiments, acryopreservation bag may have a width in a range from about 4 cm toabout 11 cm and a length in a range from about 10 cm to about 18 cm. Forexample, a cryopreservation bag may have a width in a range from about5.8 cm to about 9.8 cm and a length in a range from about 12 cm to about16 cm. In particular, an embodiment of a cryopreservation bag may have awidth of about 7.8 cm and length of about 14 cm.

Prior to use, the cryopreservation kit and/or specific componentsthereof may be sterilized. Materials used to form bags may be heatsealable. Materials for use in the bags may include, but is not limitedto polymers such as EVA, polyamides (e.g., nylons), and combinationsthereof. Open bags may be used for processing and/or disaggregationafter closing the bag using a seal and/or a clamp.

A filter may be an inline filter, a blood filter, such as a bloodadministration filter, a biological filter, and/or an in-line clumpremoval filter. The filter may be configured to remove materials fromthe processed tissue above a predetermined size to form a desiredmaterial. For example, lumps of tissue may be separated from thedisaggregated tissue using the filter. In particular, a tissuecomposition entering tubing after being filtered may have constituentshaving an average size of less than about 200 pm such that a desiredmaterial is formed. For example, the desired material may include TILs(tumor infiltrating lymphocytes) having an average size of less thanabout 170 pm.

A filter may be selected such that the processed tissue compositionentering from tubing may be enriched such that after the filter thedesired material flows into tubing in the direction of the stabilizationelement having constituents having a size in a range from about 15 pm toabout 500 μm. In some embodiments, a filter may be configured such thata tissue composition entering tubing in the direction of thestabilization element after being filtered has constituents having asize in a range from about 50 μm to about 300 pm. For example, a filtermay, in an embodiment, be configured such that a tissue compositionentering tubing after being filtered has constituents having a size in arange from about 150 μm to about 200 μm.

In some embodiments, a filter of the enrichment element may removematerials from the processed tissue outside of a predetermined sizerange from about 5 μm to about 200 pm to form a desired material. Forexample, the desired material may include TILs having an average size ina range from about 5 μm to about 200 pm. Valves may be placed apredetermined distance from a collection bag. For example, a needle freevalve may be positioned about 20 cm from a collection bag. Valves suchas needle free valves may be used to add materials to a collection bag.For example, enzyme media may be inserted into a needle free valve inorder to add the media to a collection bag. Materials to be provided viavalves include, for example, tumor digest media and/or a cryoprotectantor cryopreservation media such as DMSO and/or solutions thereof, such as55% DMSO and 5% Dextran cryopreservation media (e.g., BloodStor 55-5).

Syringes may be used to provide tumor digest media and a 55% DMSOsolution, such as 55% DMSO and 5% Dextran cryopreservation media,respectively, through needle free valves 290, 292. During processingmaterials may be selectively provided to the cryopreservation kit atpredetermined times. Further, clamps may be used to control the flow ofprovided materials such as tumor digest media and/or a cryoprotectant,such as a DMSO solution may be provided to the devices such as thecollection bag, the filter, and/or the cryopreservation bag atpredetermined times.

In some embodiments, after such a valve there may be a predeterminedamount of tubing to allow space to weld on additional components for thecryopreservation kit. For example, after some valves at least ten (10)cm of tubing may be positioned before next element. Tubing 199 may besealable and/or weldable. For example, materials for tubing may include,but is not limited to PVC (polyvinyl chloride), and/or other materialsknown in the art. In some embodiments, tubing may be sized to fitconnectors. For example, tubing may have an inner diameter in a rangefrom about 1.5 mm to about 4.5 mm and an outer diameter in a range fromabout 2.1 mm to about 6.1 mm. For example, an embodiment of acryopreservation kit may include tubing having an inner diameter in arange from about 2.9 mm to about 3.1 mm and having an outer diameter ina range from about 4.0 mm to about 4.2 mm. Tubing used incryopreservation kit 191 may vary in length with individual tubingelements having a length in a range from about 1 cm to about 30 cm.

Clamps may be used to inhibit and/or prevent movement of enzyme mediaand/or digested tissue into the filter. For example, a clamp may be usedto inhibit and/or prevent movement of enzyme media and/or digestedtissue into the filter prior to a desired filtration step. Another clamp198 inhibit and/or prevent undesired movement of the cryoprotectiveagent into the filter.

Two or more bags may be coupled together to ensure that disaggregatedproduct material may be properly stored in a particular embodiment.

In some embodiments, the invention may include an automated device forsemi-automated aseptic disaggregation, enrichment, and/or stabilizationof cells and/or cell aggregates from tissue, for example a solidmammalian tissue. An automated device for use with the invention mayinclude a programmable processor and a cryopreservation kit. In someembodiments, the cryopreservation kit may be single use. The inventionfurther relates to a semi-automatic aseptic tissue processing method.

In some embodiments, bags such as a collection bag may be used in acollection kit. Bags have an open end allowing for the addition of asample, such as a tissue sample. A connector may couple the bag totubing in a collection kit. Tubing material may be sealable and/orweldable. For example, the tubing may be sealed using energy such asheat, radio frequency, etc. The tubing material may be made from PVA.

In some embodiments, tubing may be coupled to a valve to allow additionof one or more media enzyme solutions including, but not limited tocollagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, LiberaseHI, pepsin, or mixtures thereof. For example, the valve may be a needlefree valve. Tubing used in the cryopreservation kit may include tubinghaving an outer diameter in a range from about 3.0 mm to about 5.0 mmwith an inner diameter of the tubing in a range from about 2.0 mm toabout 4 mm. In particular, tubing may have an outer diameter of4.1+/−0.1 mm and an inner diameter of about 3.0+/−0.1 mm. The length oftubing may depend on the configuration of the collection kit. Forexample, an embodiment of a collection kit may include tubing having alength in a range from about 10 cm to about 20 cm.

In some embodiments of the collection kit prototype may include one ormore clamps to inhibit and/or prevent movement of tissue and/or enzymemedia. In particular, enzyme media and/or tissue may be inhibited frommoving into a filter before a filtration step.

There are three separate elements to the treatment that may potentiallycontribute to therapeutic activity. The core elements are TILs, such asUTILs, which have the potential to eliminate tumor cells by a variety ofmechanisms utilized by T-cells as part of their normal function.

These mechanisms include: direct cytotoxicity by [a] releasingcytotoxins (e.g. perforin, granzymes, and granulysin), which entertarget cells by close engagement and induce cell death; and by [b]cell-surface interactions between T cell and target such as binding FASLigand mediated cytotoxicity inducing apoptosis; and indirect methods(e.g. cytokine production) that have the ability to recruit andstimulate secondary effector cells to engage and induce tumor celldeath.

TILs, in particular UTILs, are an autologous product; consequently, eachbatch manufactured provides a single dose for a specified patient. Thereare no sub-batches or pooling of batches. The drug product is a smallaseptically prepared batch of T cells (5×10⁹ to 5×10¹⁰) cryopreserved ina saline based solution with 8.5% human serum albumin and 10% DMSO ofbetween 125-270 mL for a single intravenous infusion after thawing.

There are several advantages in the present invention as compared toU.S. Pat. No. 10,398,734 (“the '734 patent”). The first step in the '734patent is transforming the tumor bulk into fragments from which TILs arecultured. In contrast, the present invention liberates TILs from thetumor, which was preserved and disaggregated under aseptic conditionsfollowing resection in the aseptic kit, from which a cell suspension isprepared, and cryopreserves the resulting TILs by freezing. The presentinvention provides a diverse population of TILs representing thediversity that exists inside the tumor. And because they are ahomogenous suspension, the TILs that are expanded in the culture willretain that diversity, which gives the greatest chance of addressing thedi verse population of cancer cells that reside within the tumor.

In contrast, the manufacturing process of the '734 patent starts withfragments of tissue that have already experienced deterioration of theinternal cell population during shipping and any further delay beforestarting processing. In addition, TILs used for manufacturing will onlybe TIL that expand from the tissue fragments and not any TIL that areretained in the interior, so that the resulting cell population may notreflect the full diversity of tumor environment.

Another difference is that the entry into closed manufacturingprocessing occurs much sooner and with less chance of contamination inthe process of the present invention than in the process of the '734patent. In particular, the disruption of the tumor tissue occurs in aclosed processing system in the present application, rather than theextensive fragmentation process which the '734 patent describes asoccurring in an open operation in a biological safety cabinet.

Because the starting material for the present invention is preservedunder aseptic conditions in the aseptic kit, the full manufacturingprocess, which can be run on a cryopreserved tumor cell suspension, canbe scheduled and run at high capacity and efficiency. In contrast,because the '734 patent starts with unfrozen tissue, the fragmentationand “growth-out” steps are run on a stand-by basis with lower efficiencyof capacity utilization. Removing this intermediate freezing step, inthe '734 patent, shortens the manufacturing process overall, but meansthat the entire process is run on a stand-by basis, meaning thatmanufacturing down time has significant consequences to themanufacturing facility of the '734 patent as there cannot be any delaysand planning a down period for manufacturing requires will require allproducts in process to be completed and new surgeries to be stopped.

The advantage of the process of the present application is that tissue,in the form of a resected tumor, can be collected in advance of arequirement for TIL therapy, transported, processed, cryopreserved andstored in the aseptic kit until and if manufacturing is needed sopatients with earlier stage disease can be harvested and stored whilethey have alternative therapies. Consequently, there is little or noimpact upon the timing or geolocation of tumor collection and subsequentmanufacturing. Whereas in the '734 patent, this is not possible and fullmanufacturing of a drug product has to occur before cells can be frozenand held.

As mentioned above, these are very different culture processes that willgenerate different populations of cells from which to initiate the REPculture, as reflected in the very different numbers of cells needed toseed the REP culture, 1-20 million (the present invention) versus 25-200million (the '734 patent). In the present invention during the initialTIL expansion the culture seeding uses a cell suspension (i.e. cellsthat grow out of the disaggregated and cryopreserved cells which will bea mixture of resident and emergent T cells) versus outgrowth from thechunks (i.e. emergent cells); this means the REP is not just seeded withemergent T cells. In addition, the present invention can utilize bothsolid and flexible closed containers where flexible containers enable amore optimal environment based on the amount of tumor suspension derivedrather than a number of chunks as defined in the '734 patent].

Metastatic tumor material is surgically removed using standard surgicalpractice within a surgical operating room. Prior to disaggregationextraneous material is removed (i.e. non-tumor material as definedmacroscopically) and the tumor material is transferred into a sterilebag.

The following may be involved in tumor starting material acceptancetesting. First, the source tissue is confirmed to be tumor material.Second, a representative sample of the disaggregated tissue is assessedfor microbial load and where present antibiotic sensitivities defined(manufacturing may be performed at risk with antibiotics) but finalmaterial must be negative for microbial growth. Third, quantity andviability of TIL and tumor cells can be assessed by flow cytometry.

The methods of the invention comprise the step of asepticallydisaggregating a tumor resected from a subject thereby producing adisaggregated tumor, wherein the resected tumor is sufficientlydisaggregated if it can be cryopreserved without cell damage. In anadvantageous embodiment, a programmable processor of a semi-automaticdevice may control disaggregation enabling the surfaces withindisaggregation flexible containers to mechanically crush and shear thesolid tissue (see, e.g., PCT Publication No. WO 2018/130845).Disaggregation surfaces may be controlled, for example, by mechanicalpistons.

For enzymatic digestion, a cell suspension (containing both T cells andtumor cells) is generated from the resected metastatic tumor using anenzyme mixture of DNase 1 and Collagenase (Type IV). The combination ofthe repeated mechanical compression exposes additional surfaces for theenzymes to access and the enzymatic reaction speed up the process ofturning a solid tissue into a cell suspension prior to optionalcryopreservation. In one embodiment upon completion of thedisaggregation step a DMSO based cryoprotectant is added just prior to acontrolled rate freezing cycle. In some embodiments, the enzymaticbreakdown of the solid tissue may be by the selection and provision ofone or more media enzyme solutions such as collagenase, trypsin, lipase,hyaluronidase, deoxyribonuclease, Liberase HI, pepsin, or any mixturethereof. Enzymatic digestion of the resected metastatic tumor can occurin the disaggregation flexible containers of the semi-automatic device.

By way of example, in another embodiment of the method of the invention,where the disaggregation process is being supplemented with enzymaticdigestion the media formulation for enzymatic digestion must besupplemented with enzymes that aid in protein breakdown causing the cellto cell boundaries to break down.

Various liquid formulations known in the art of cell culturing or cellhandling can be used as the liquid formulation used for celldisaggregation and enzymatic digestion of solid tissues, including butnot limited to one or more of the following media Organ PreservationSolutions, selective lysis solutions, PBS, DMEM, HBSS, DPBS, RPMI,Iscove's medium, XVIVO™, AIM-Vim, Lactated Ringer's solution, Ringer'sacetate, saline, PLASMALYTE™ solution, crystalloid solutions and IVfluids, colloid solutions and IV fluids, five percent dextrose in water(D5W), Hartmann's SolutionDMEM, HBSS, DPBS, RPMI, AIM-V™, Iscove'smedium, XVIVO™, each can be optionally supplemented with additional cellsupporting factors e.g. with fetal calf serum, human serum or serumsubstitutes or other nutrients or cytokines to aid in cell recovery andsurvival or specific cell depletion. The media can be standard cellmedia like the above mentioned media or special media for e.g. primaryhuman cell culture (e.g. for endothelia cells, hepatocytes orkeratinocytes) or stem cells (e.g. dendritic cell maturation,hematopoietic expansion, keratonocytes, mesenchymal stem cells or Tcells). The media may have supplements or reagents well known in theart, e.g. albumins and transport proteins, amino acids and vitamins,metal-ion(s), antibiotics, attachments factors, de-attachment factors,surfactants, growth factors and cytokines, hormones or solubilizingagents. Various media are commercially available e.g. from ThermoFisher,Lonza, or Sigma-Aldrich or similar media manufacturers and suppliers.

The liquid formulation required for enzymatic digestion must havesufficient calcium ions present in the of at least 0.1 mM up to 50 mMwith an optimal range of 2 to 7 mM ideally 5 mM.

The solid tissue to be digested can be washed after disaggregation witha liquid formulation containing chelating agents EGTA and EDTA to removeadhesion factors and inhibitory proteins prior to washing and removal ofEDTA and EGTA prior to enzymatic digestion.

The liquid formulation required for enzymatic digestion is more optimalwith minimal chelating agents EGTA and EDTA which can severely inhibitenzyme activity by removing calcium ions required for enzyme stabilityand activity. In addition, β-mercaptoethanol, cysteine and8-hydroxyquinoline-5-sulfonate are other known inhibitory substances.

Processing of tumor material using dissection, enzymatic digestion andhomogenization produces a single cell suspension of TILs, in particularUTILs, which can be directly cryopreserved to stabilize the startingmaterial for subsequent processing via the first expansion of the cellsuspension of TILs, in particular UTILs, in IL-2 to obtain a firstpopulation of TILs, in particular UTILs.

The methods also comprise the step of cryopreserving the disaggregatedtumor, e.g. the cell suspension. Cryopreserving the disaggregated tumoris carried out on the same day as carrying out the step of asepticallydisaggregating a tumor resected from a subject thereby producing adisaggregated tumor, wherein the resected tumor is sufficientlydisaggregated if it can be cryopreserved without cell damage. Forexample, cryopreserving is carried out 5, 10, 20, 30, 40, 50, 60, 70,80, or 90 minutes, or 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 hoursfollowing the step of disaggregating the tumor. Cryopreservation of thedisaggregated tumor, as a single cell suspension obtained from theenzymatic disaggregation in the disaggregation module of thesemi-automatic device, is carried out by cooling and/or maintaining thesuspension at a temperature between 8° C. and at least −80° C. or below.Disaggregation could be as quick as 5 mins but most usually 45 mins to 1hour and the cryopreservation can be a quick as 60 mins or up to 150mins. In one embodiment, the methods include storing the cryopreserveddisaggregated tumor. As described in preferred embodiments, the devicecomprises at least one cell container for cryopreservation wherein thecontainers are a flexible container manufactured from resilientdeformable material. In this embodiment of the device, the finalcontainer is either transferred directly to a freezer −20 to −190° C. ormore optimally located in the controlled rate freezing apparatus eitherassociated with the device or supplied separately (manufactured by forexample Planer Products or Asymptote Ltd) in which the temperature ofthe freezing chamber and the flexible storage container(s) employed tocontain the enriched disaggregated solid tissue container is controlledeither by: injecting a cold gas (normally nitrogen for example Planerproducts); or by removing heat away from the controlled coolingsurface(s). Both methods result in the ability to accurately controlwith an error of less than 1° C. or more preferable 0.1° C. the freezingprocess at the required rate for the specific cell(s) to be frozen basedon the freezing solution and the desired viability of the product. Thiscryopreservation process must take into account the ice nucleationtemperature which is ideally as close as possible to the meltingtemperature of the freezing solution. Followed by crystal growth in anaqueous solution, water is removed from the system as ice, and theconcentration of the residual unfrozen solution increases. As thetemperature is lowered, more ice forms, decreasing the residualnon-frozen fraction which further increases in concentration. In aqueoussolutions, there exists a large temperature range in which ice co-existswith a concentrated aqueous solution. Eventually through temperaturereduction the solution reaches the glass transition state at which pointthe freezing solution and cells move from a viscous solution to asolid-like state below this temperature the cells can undergo no furtherbiological changes and hence are stabilized, for years potentiallydecades, until required.

Ice nucleation and crystal growth involves release of heat to thefreezing solution and the cellular microenvironment and it is desirableto maintain cooling of cells and freezing solution even as the freezingfluid resists temperature changes while undergoing phase change.Depending on whether disaggregation includes enzymatic disaggregation,and what is the optimal temperature of enzymatic digestion for a givenenzyme, enzyme concentration and tissue type, temperatures at the startof cryopreservation include, without limitation, 40° C., 39° C., 38° C.,37° C., 36° C., 35° C., 34° C., 33° C., 32° C., 31° C., 30° C., 29° C.,28° C., 27° C., 26° C., 25° C., 24° C., 23° C., 22° C., 21° C., and 20°C., i.e., temperatures ranging from a mammalian body temperature to roomtemperature, and further include lower refrigeration temperatures suchas, without limitation, 10° C., 8° C., 6° C., 5° C., 4° C., 3° C., and2° C. Target temperatures for cryogenic cooling include, withoutlimitation, −60° C., −65° C., −70° C., −75° C., −80° C., −85° C., −90°C., and temperatures in between as well as colder temperatures down tothe temperature of liquid nitrogen vapor storage (−195.79° C.). Incertain embodiments, the methods and devices used according to theinvention are designed or programmed to minimize the time fromphysiological temperature or digestion temperature to cryostoragetemperature. In certain embodiments, the methods and devices usedaccording to the invention for cryopreservation are advantageouslydesigned and programmed for cooling under conditions whereby heatrelease to, into, around or in an environment including cells, as mediacrystalizes, is minimized or avoided. In certain embodiments, methodsare designed and/or devices programmed for continuous cooling fromdisaggregation temperature down to a cryogenic target temperature.Exemplary programmed cooling rates include, without limitation, −0.5°C./min, −1° C./min, −1.5° C./min, −2° C./min, or −2.5° C./min. Thecooling rates are program targets and may vary over a cooling cycle. Thecooling rates may vary, for example by ±0.1° C./min, ±0.2° C./min, 0.3°C./min, ±0.4° C./min, or ±0.5° C./min. In an embodiment of theinvention, the cryopreservation temperature is −80° C.±10° C. and thedevice is programmed to reduce temperature by 1° C./min or 1.5° C./minor 2° C./min or 1° C./min±0.5° C./min or 1.5° C./min±0.5° C./min or 2°C./min±0.5° C./min.

In some embodiments, the present methods provide for obtaining youngTILs, which are capable of increased replication cycles uponadministration to a subject/patient and as such may provide additionaltherapeutic benefits over older TILs (i.e., TILs which have furtherundergone more rounds of replication prior to administration to asubject/patient). Features of young TILs have been described in theliterature, for example Donia, at al., Scandinavian Journal ofImmunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res,16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005);Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., JImmunother 32:415-423 (2009); Robbins, et al., J Immunol 2004;173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou, etal., J Immunother, 28:53-62 (2005); and Tran, et al., J Immunother,31:742-751 (2008), all of which are incorporated herein by reference intheir entireties.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained by thepresent method exhibit an increase in the T-cell repertoire diversity ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide. In some embodiments, the TILs obtained bythe present method exhibit an increase in the T-cell repertoirediversity as compared to freshly harvested TILs and/or TILs. In someembodiments, the TILs obtained in the first expansion exhibit anincrease in the T-cell repertoire diversity. In some embodiments, theincrease in diversity is an increase in the immunoglobulin diversityand/or the T-cell receptor diversity. In some embodiments, the diversityis in the immunoglobulin is in the immunoglobulin heavy chain. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin light chain. In some embodiments, the diversity is in theT-cell receptor. In some embodiments, the diversity is in one of theT-cell receptors selected from the group consisting of alpha, beta,gamma, and delta receptors. In some embodiments, there is an increase inthe expression of T-cell receptor (TCR) alpha and/or beta. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) alpha. In some embodiments, there is an increase in the expressionof T-cell receptor (TCR) beta. In some embodiments, there is an increasein the expression of TCRab (i.e., TCRαβ).

The methods of the invention also comprise the step of performing afirst expansion by culturing the disaggregated tumor in a cell culturemedium comprising IL-2 to produce a first population of TILs, inparticular UTILs. The cells resulting from the steps described above arecultured in serum containing IL-2 under conditions that favor the growthof TILs over tumor and other cells. In some embodiments, the tumordigests are incubated in 2 mL wells in media comprising inactivatedhuman AB serum with 6000 IU/mL of IL-2. This primary cell population iscultured for a period of days, generally from 3 to 14 days, resulting ina bulk TIL population, generally about 1×10⁸ bulk TIL cells. In someembodiments, this primary cell population is cultured for a period of 7to 14 days, resulting in a bulk TIL population, generally about 1×10⁸bulk TIL cells. In some embodiments, this primary cell population iscultured for a period of 10 to 14 days, resulting in a bulk TILpopulation, generally about 1×10⁸ bulk TIL cells. In some embodiments,this primary cell population is cultured for a period of about 11 days,resulting in a bulk TIL population, generally about 1×10⁸ bulk TILcells.

In a preferred embodiment, expansion of TILs may be performed using aninitial bulk TIL expansion step as described below and herein, followedby a second expansion (including rapid expansion protocol (REP) stepsand followed by restimulation REP steps) as described below and herein.

In an advantageous embodiment, the cryopreserved disaggregated tumortissue is thawed and resuspended 1:9 in T cell media (T cell culturemedia contract manufactured for Immetacyte supplemented with thefollowing additives 10% FBS and 3000 IU/mL IL-2) prior to filtrationthrough an inline 100-270 μm filter and centrifugation in a 50 mLcentrifuge tube prior to resuspension in 20 mL. A sample may be takenfor flow cytometry analysis to quantify a number of HLA-A, B, C andCD58+, and DRAQ7− cells. In some embodiments this may be seeded using analternative manual (such as but not limited to a haemocytometer) oralternative automated total viable cell counting device such as but notlimited to NucleoCounter™; Guava®; automated blood analysis and counter;pipette based cell counter such as but not limited to Scepter™.

In one embodiment, resuspended cryopreserved disaggregated tumor tissueis cultured in serum containing IL-2 under conditions that favor thegrowth of TILs over tumor and other cells. In some embodiments, thetumor digests are incubated in 2 mL wells in media comprisinginactivated human AB serum (or, in some cases, as outlined herein, inthe presence of an artificial antigen-presenting [aAPC] cell population)with 6000 IU/mL of IL-2. This primary cell population is cultured for aperiod of days, generally from 10 to 14 days, resulting in a bulk TILpopulation, generally about 1×10⁸ bulk TIL cells. In some embodiments,the growth media during the first expansion comprises IL-2 or a variantthereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2).In some embodiments the IL-2 stock solution has a specific activity of20-30×10⁶ IU/mg for a 1 mg vial. In some embodiments the IL-2 stocksolution has a specific activity of 20×106 IU/mg for a 1 mg vial. Insome embodiments the IL-2 stock solution has a specific activity of25×10⁶ IU/mg for a 1 mg vial. In some embodiments the IL-2 stocksolution has a specific activity of 30×10⁶ IU/mg for a 1 mg vial. Insome embodiments, the 1L-2 stock solution has a final concentration of4-8×10⁶ IU/mg of IL-2. In some embodiments, the IL-2 stock solution hasa final concentration of 5-7×10⁶ IU/mg of IL-2. In some embodiments, theIL-2 stock solution has a final concentration of 6×10⁶ IU/mg of IL-2. Insome embodiments, the first expansion culture media comprises about10,000 IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL ofIL-2, about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000IU/mL of IL-2. In some embodiments, the first expansion culture mediacomprises about 9,000 IU/mL of IL-2 to about IU/mL of IL-2. In someembodiments, the first expansion culture media comprises about 8,000IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments, thefirst expansion culture media comprises about 7,000 IU/mL of IL-2 toabout 6,000 IU/mL of 1L-2. In some embodiments, the first expansionculture media comprises about 6,000 IU/mL of IL-2. In an embodiment, thecell culture medium further comprises IL-2. In some embodiments, thecell culture medium comprises about 3000 IU/mL of IL-2. In anembodiment, the cell culture medium further comprises IL-2. In apreferred embodiment, the cell culture medium comprises about 3000 IU/mLof IL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.

In some embodiments, first expansion culture media comprises about 500IU/mL of IL-12, about 400 IU/mL of IL-12, about 300 IU/mL of IL-12,about 200 IU/mL of IL-12, about 180 IU/mL of IL-12, about 160 IU/mL ofIL-12, about 140 IU/mL of IL-12, about 120 IU/mL of IL-12, or about 100IU/mL of IL-12. In some embodiments, the first expansion culture mediacomprises about 500 IU/mL of IL-12 to about 100 IU/mL of IL-12. In someembodiments, the first expansion culture media comprises about 400 IU/mLof IL-12 to about 100 IU/mL of IL-12. In some embodiments, the firstexpansion culture media comprises about 300 IU/mL of IL-12 to about 100IU/mL of IL-12. In some embodiments, the first expansion culture mediacomprises about 200 IU/mL of IL-12. In some embodiments, the cellculture medium comprises about 180 IU/mL of 1L-12. In an embodiment, thecell culture medium further comprises 1L-12. In a preferred embodiment,the cell culture medium comprises about 180 IU/mL of IL-12.

In some embodiments, first expansion culture media comprises about 500IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the first expansion culture media comprises about 400 IU/mLof IL-15 to about 100 IU/mL of IL-15. In some embodiments, the firstexpansion culture media comprises about 300 IU/mL of IL-15 to about 1001U/mL of 1L-15. In some embodiments, the first expansion culture mediacomprises about 200 IU/mL of IL-15. In some embodiments, the cellculture medium comprises about 180 IU/mL of IL-15. In an embodiment, thecell culture medium further comprises IL-15. In a preferred embodiment,the cell culture medium comprises about 180 IU/mL of IL-15.

In some embodiments, first expansion culture media comprises about 500IU/mL of IL-18, about 400 IU/mL of IL-18, about 300 IU/mL of IL-18,about 200 IU/mL of IL-18, about 180 IU/mL of IL-18, about 160 IU/mL ofIL-18, about 140 IU/mL of IL-18, about 120 IU/mL of IL-18, or about 100IU/mL of IL-18. In some embodiments, the first expansion culture mediacomprises about 500 IU/mL of IL-18 to about 100 IU/mL of IL-18. In someembodiments, the first expansion culture media comprises about 400 IU/mLof IL-18 to about 100 IU/mL of IL-18. In some embodiments, the firstexpansion culture media comprises about 300 IU/mL of IL-18 to about 100IU/mL of IL-18. In some embodiments, the first expansion culture mediacomprises about 200 IU/mL of IL-18. hi some embodiments, the cellculture medium comprises about 180 IU/mL of IL-18. In an embodiment, thecell culture medium further comprises IL-18. In a preferred embodiment,the cell culture medium comprises about 180 IU/mL of IL-18.

In some embodiments, first expansion culture media comprises about 20IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, orabout 0.5 IU/mL of IL-21. In some embodiments, the first expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the first expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of 1L-21. In some embodiments, the firstexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the first expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

Also contemplated for the culture media are combinations ofinterleukins, such as but not limited to, IL-2, 1L-12, 1L-15, 1L-18 and1L-21. Other cytokines are also contemplated, such as IL-23, IL-27,IL-35, IL-39, IL-18, IL-36, IL-37, IL-38, IFN-alpha, IFN-beta, IFN-gammaor a combination thereof along with IL-2, IL-12, IL-15, IL-18 and IL-21.Antibodies, such as Th2 blocking reagents, are also contemplated, suchas but not limited to, IL-4 (aIL4), anti-IL-4 (aIL4R), anti-IL-SR(aIL5R), anti-IL-5 (aILS), anti-IL13R (aIL13R), or anti-IL13 (aIL13).

In some embodiments, the first TIL expansion can proceed for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, or 14 days. In some embodiments, the firstTIL expansion can proceed for 1 day to 14 days. In some embodiments, thefirst TIL expansion can proceed for 2 days to 14 days. In someembodiments, the first TIL expansion can proceed for 3 days to 14 days.In some embodiments, the first TIL expansion can proceed for 4 days to14 days. In some embodiments, the first TIL expansion can proceed for 5days to 14 days. In some embodiments, the first TIL expansion canproceed for 6 days to 14 days. In some embodiments, the first TILexpansion can proceed for 7 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 8 days to 14 days. In someembodiments, the first TIL expansion can proceed for 9 days to 14 days.In some embodiments, the first TIL expansion can proceed for 10 days to14 days. In some embodiments, the first TIL expansion can proceed for 11days to 14 days. In some embodiments, the first TIL expansion canproceed for 12 days to 14 days. In some embodiments, the first TILexpansion can proceed for 13 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 14 days. In some embodiments, thefirst TIL expansion can proceed for 1 day to 11 days. In someembodiments, the first TIL expansion can proceed for 2 days to 11 days.In some embodiments, the first TIL expansion can proceed for 3 days to11 days. In some embodiments, the first TIL expansion can proceed for 4days to 11 days. In some embodiments, the first T1L expansion canproceed for 5 days to 11 days. In some embodiments, the first TILexpansion can proceed for 6 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 7 days to 11 days. In someembodiments, the first TIL expansion can proceed for 8 days to 11 days.In some embodiments, the first TIL expansion can proceed for 9 days to11 days. In some embodiments, the first TIL expansion can proceed for 10days to 11 days. In some embodiments, the first TIL expansion canproceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the first expansion. In someembodiments, 1L-2, 1L-7, 1L-15, and/or IL-21 as well as any combinationsthereof can be included during the first expansion. In some embodiments,a combination of IL-2, IL-15, and IL-21 are employed as a combinationduring the first expansion.

In some embodiments, the first expansion is performed in a closed systembioreactor. In some embodiments, a closed system is employed for the TILexpansion, as described herein. In some embodiments, a single bioreactoris employed. In some embodiments, the single bioreactor employed isexample a G-REX-10 or a G-REX-100 or advantageously the device of WO2018/130845. In some embodiments, the closed system bioreactor is asingle bioreactor.

Advantageously, the TIL population obtained from the first expansion,referred to as the second TIL population, can be subjected to a secondexpansion (which can include expansions sometimes referred to as REP.Similarly, in the case where genetically modified TILs will be used intherapy, the first TIL population (sometimes referred to as the bulk TILpopulation) or the second TIL population (which can in some embodimentsinclude populations referred to as the REP TIL populations) can besubjected to genetic modifications for suitable treatments prior toexpansion or after the first expansion and prior to the secondexpansion.

Lentiviruses are efficient gene transfer vehicles due to their abilityto transduce both dividing and nondividing cells. While the mostthoroughly investigated of the lentiviral gene therapy vectors arederived from human immunodeficiency virus (HIV) type 1, gene therapyvectors based on other primate and non-primate lentiviruses have alsobeen developed, including, HIV-2, SW, feline immunodeficiency virus(Hy), equine infectious anemia virus (EIAV), caprine arthritisencephalitis virus (CAEV), visna virus, and Jembrana disease virus(JDV).

Replication-deficient viral vectors are essential in preventinginfection of a patient with a potentially deadly virus. Lentiviralvectors have have been developed to become safer and more efficient.Recent third-generation vectors removed all accessory genes that aid invirulence and pathogenicity while splitting the remaining genes, whichare vital for expression of a transgene across three plasmids. See,e.g., U.S. Patent Publication 2006/0024274.

EIAV gene transfer vectors were shown to be effective in transducingproliferating and Gi-arrested cells in vitro. Mitrophanous, et al.,1999. Stable gene transfer to the nervous system using a non-primatelentiviral vector. Gene Ther. 6: 1808-1818; Olsen, J. C., 1998, Genetransfer vectors derived from equine infectious anemia virus. Gene Ther.5: 1481-1487; Olsen, J. C., 2001, E1AV, CAEV and Other Lentivirus VectorSystems, Somat Cell Mol Genet, Vol. 26, Nos. 1/6, 131-45.

Heemskerk, B. et al., 2008, Adoptive cell therapy for patients withmelanoma, using tumor-infiltrating lymphocytes genetically engineered tosecrete interleukin-2. Human gene therapy, 19(5), 496-510, describesTILs genetically engineered to express IL-2 to prolong TIL survival.Patient TIL was transfected during a first expansion with a retroviralvector based on Moloney murine leukemia virus (MMLV) followed by asecond expansion to obtain sufficient numbers for treatment.

In brief, the SBIL2 vector, containing the MFG backbone derived fromMoloney murine leukemia virus (MMLV) with a cDNA copy of the human IL-2gene under the control of the 5′ long terminal repeat (LTR) promoter,was pseudotyped in the PG13 packaging cell line, which provides thegibbon ape leukemia virus (GaLV) envelope protein. A stable producerclone (PGI3SBIL2#3) was generated that contained three copies of theintegrated retroviral IL-2 DNA. Clinical GMP-grade SBIL2 retroviralsupernatant was produced by the National Gene Vector Laboratory atIndiana University (Indianapolis, IN). For TIL transduction, 6-wellnon-tissue-culture plates (Becton Dickinson, Franklin Lakes, NJ) werecoated with Retronectin (CH-296, 25 μg/ml in phosphate-buffered saline[PBS], GMP grade; Takara Bio, Otsu, Japan), blocked with PBS-2% humanserum albumin (HSA), and preloaded for 4 hr with thawed SBIL2 viralsupernatant (5 ml/well) at 32° C. and 10% CO2. TILs were added at 3nil/well for 18-24 hr at 37° C. and 5% CO2, transferred to a second setof SBIL2-loaded plates, and cultured for an additional 18-24 hr, afterwhich TILs were harvested and resuspended in fresh medium.

Zhang, L. et al., 2015, Tumor-infiltrating lymphocytes geneticallyengineered with an inducible gene encoding interleukin-12 for theimmunotherapy of metastatic melanoma, Clinical Cancer Research 21(10),2278-2288. describes TILs genetically engineered to secrete IL-12selectively at a tumor site. TILs were transduced with a MSGV1γ-retroviral vector carrying a gene encoding a single-chain IL-12 drivenby a nuclear factor of activated T cells (NFAT) promoter. activated Tcells promoter.

MSGV-1 is derived from the MSGV vector that utilizes the murine stemcell virus long terminal repeat and contains an extended gag region andKozak sequence. The gene encoding human single chain IL-12 wassynthesized with the order IL-12 p40, linker G6S and IL-12 p35 driven byan NFAT responsive promoter and inserted into the MSGV-1 vector reverseto the 5′ LTR direction. A high-titer PG13 cell based producer cell linewas generated and retroviral supernatant was produced by the NCI SurgeryBranch Vector Production Facility (Bethesda, MD) under goodmanufacturing practice (GMP) conditions. The vector supernatant wastested and passed all currently required US Food and Drug Administrationguidelines for the production of recombinant gamma-retroviral vectorsfor clinical application.

The transduction procedure was initiated by stimulatingtumor-infiltrating lymphocytes (TILs) with 30 ng/ml anti-CD3 mAbOrthoclone OKT3 (Centocor Ortho Biotech, Raritan, NJ), 3000 IU/mlrecombinant human IL-12 and 4 Gy irradiated allogeneic PBMC feeder cellsat a ratio of 200 feeder cells for every TIL. Cells were harvested fortransduction on day 4 and/or day 5 using RetroNectin (CH-296; Takara BioInc., Otsu, Japan) coated non-tissue culture 6-well plates. Vectorsupernatant was “spin loaded” onto coated plates by centrifugation at2000 g for 2 hours at 32° C. Retroviral vector supernatant was aspiratedfrom the wells and 2×10⁶ stimulated TIL cells were added each wellfollowed by centrifugation at 1000 g for 10 minutes. Plates wereincubated at 37° C. overnight and cells were harvested for the 2ndtransduction the following day. Cells for the first 21 patientsunderwent two transductions. Cells for patients 12 underwent only onetransduction.

Jones, S. et al., 2009, Lentiviral vector design for optimal T cellreceptor gene expression in the transduction of peripheral bloodlymphocytes and tumor-infiltrating lymphocytes. Human gene therapy,20(6), 630-640, describes development of promoters for use in lentiviralvectors to express genes in transduced T lymphocytes and constructeffective antitumor T cells.

TILs were obtained from surgical specimens. PBLs were thawed from frozenstock stored at −180° C. and placed into culture in AIM-V andinterleukin-2 (1L-2; Cetus, Emeryville, CA) at 300 IU/ml. For OKT3stimulation, the cells were either initially place in medium withanti-CD3 antibody, OKT3 (Ortho Biotech, Bridgewater, NJ) at 50 ng/ml, orwere placed in OKT3 medium after transduction, at the initial changingof the culture medium. For transduction of the PBLs or TILs, 1×106 cellswere adjusted to a final volume of 1 ml in a 24-well tissueculture-treated plate with the viral supernatant and Polybrene (finalconcentration, 8 m/ml). The cells were transduced by centrifugation ofthe plates for 1.5 hr at 1000× g, 32° C. The plates were placed in a 37°C., humidified 5% CO2 incubator overnight, and the medium was replacedthe next day. TILs were subject to the rapid expansion protocol (REP) aspreviously described, using OKT3 (50 ng/ml), 1L-2 (5000 Ill/m1), andirradiated allogeneic peripheral blood mononuclear cells from threedifferent donors (TIL:feeder ratio, 1:100). Six days post-REP, TILs weretransduced as described and returned to culture.

Beane, J. D. et al., 2015, Clinical Scale Zinc Finger Nuclease-mediatedGene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatmentof Metastatic Melanoma. Molecular therapy: 23(8), 1380-1390 describesclinical scale gene editing of PD-1 by electroporation of mRNA encodingPD-1 specific zinc finger nuclease (ZFN)-mediated gene editing.

In order to generate a sufficient number of transduced T cells foradoptive cell transfer, the TIL were induced to proliferate using aREP.46 Briefly, 1×10⁷ TIL were combined with 1×10⁹ allogeneic,irradiated (5,000 rad) peripheral blood mononuclear cells (PBMC), andthese cells were suspended in 400 ml of T-cell media containing 30 ng/mlof OKT3. The cells were cultured in a G-Rex100 flask at 37° C. and 5%CO2. Five days later, 200 ml of media was aspirated and replaced. Sevendays after the start of the REP, TIL were harvested and washed two timeswith Hyclone Electroporation Buffer (Hyclone Laboratories, Logan, UT).Cells were then counted and resuspended in electroporation buffer at aconcentration of 1×108/ml. Cells were then transferred to the MaxCyteCL-2 processing assembly and mixed with 120 μg/ml of PD-1 ZFN mRNA (orGFP mRNA for GFP transfected TIL/GFP). Electroporation was performed asper MaxCyte's protocol. Following electroporation, TIL were transferredfrom the processing assembly to a T-175 flask and placed in an incubatorat 37° C. for 20 minutes. Following this incubation step, TIL wereresuspended in AIM-V media at a concentration of 1×10⁶/ml. Cells werethen placed in an incubator set at 30° C. for an overnight lowtemperature incubation as previously described. The following day, TILwere transferred to a 37° C. incubator and left undisturbed until REPday 10 (3 days following electroporation).

In some embodiments, the TILs obtained from the first expansion arestored until phenotyped for selection. In some embodiments, the TILsobtained from the first are not stored and proceed directly to thesecond expansion. Thus, the methods comprise the step of performing asecond expansion by culturing the first population of TILs, inparticular UTILs, with additional IL-2, OKT-3, and antigen presentingcells (APCs), to produce a second population of TILs. In someembodiments, the TILs obtained from the first expansion are notcryopreserved after the first expansion and prior to the secondexpansion. In some embodiments, the transition from the first expansionto the second expansion occurs at about 3 days, 4, days, 5 days, 6 days,7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 daysafter the cryopreserved disaggregated tumor tissue is thawed. In someembodiments, the transition from the first expansion to the secondexpansion occurs at about 3 days to 21 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs atabout 4 days to 14 days after the cryopreserved disaggregated tumortissue is thawed. In some embodiments, the transition from the firstexpansion to the second expansion occurs at about 4 days to 10 daysafter the cryopreserved disaggregated tumor tissue is thawed. In someembodiments, the transition from the first expansion to the secondexpansion occurs at about 7 days to 14 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs atabout 14 days after the cryopreserved disaggregated tumor tissue isthawed. In some embodiments the seeding of the REP culture occurs 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 daysafter the cryopreserved disaggregated tumor tissue is thawed.

In some embodiments, the transition from the first expansion to thesecond expansion occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14days after the cryopreserved disaggregated tumor tissue is thawed. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 1 day to 14 days after the cryopreserved disaggregatedtumor tissue is thawed. In some embodiments, the first TIL expansion canproceed for 2 days to 14 days. In some embodiments, the transition fromthe first expansion to the second expansion occurs 3 days to 14 daysafter the cryopreserved disaggregated tumor tissue is thawed. In someembodiments, the transition from the first expansion to the secondexpansion occurs 4 days to 14 days after the cryopreserved disaggregatedtumor tissue is thawed. In some embodiments, the transition from thefirst expansion to the second expansion occurs 5 days to 14 days afterthe cryopreserved disaggregated tumor tissue is thawed. In someembodiments, the transition from the first expansion to the secondexpansion occurs 6 days to 14 days after the cryopreserved disaggregatedtumor tissue is thawed. In some embodiments, the transition from thefirst expansion to the second expansion occurs 7 days to 14 days afterthe cryopreserved disaggregated tumor tissue is thawed. In someembodiments, the transition from the first expansion to the secondexpansion occurs 8 days to 14 days after the cryopreserved disaggregatedtumor tissue is thawed. In some embodiments, the transition from thefirst expansion to the second expansion occurs 9 days to 14 days afterthe cryopreserved disaggregated tumor tissue is thawed. In someembodiments, the transition from the first expansion to the secondexpansion occurs 10 days to 14 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs 11days to 14 days after the cryopreserved disaggregated tumor tissue isthawed. In some embodiments, the transition from the first expansion tothe second expansion occurs 12 days to 14 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs 13days to 14 days after the cryopreserved disaggregated tumor tissue isthawed. In some embodiments, the transition from the first expansion tothe second expansion occurs 14 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs 1 dayto 11 days after the cryopreserved disaggregated tumor tissue is thawed.In some embodiments, the transition from the first expansion to thesecond expansion occurs 2 days to 11 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs 3days to 11 days after the cryopreserved disaggregated tumor tissue isthawed. In some embodiments, the transition from the first expansion tothe second expansion occurs 4 days to 11 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs 5days to 11 days after the cryopreserved disaggregated tumor tissue isthawed. In some embodiments, the transition from the first expansion tothe second expansion occurs 6 days to 11 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs 7days to 11 days after the cryopreserved disaggregated tumor tissue isthawed. In some embodiments, the transition from the first expansion tothe second expansion occurs 8 days to 11 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs 9days to 11 days after the cryopreserved disaggregated tumor tissue isthawed. In some embodiments, the transition from the first expansion tothe second expansion occurs 10 days to 11 days after the cryopreserveddisaggregated tumor tissue is thawed. In some embodiments, thetransition from the first expansion to the second expansion occurs 11days after the cryopreserved disaggregated tumor tissue is thawed.

In some embodiments, the TILs are not stored after the first expansionand prior to the second expansion, and the TILs proceed directly to thesecond. In some embodiments, the transition occurs in closed system, asdescribed herein. In some embodiments, the TILs from the firstexpansion, the second population of TILs, proceeds directly into thesecond expansion with no transition period.

In some embodiments, the transition from the first expansion to thesecond expansion is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100 or Xuri WAVE bioreactor. In some embodiments,the closed system bioreactor is a single bioreactor.

In some embodiments, the TIL cell population is expanded in number afterharvest and initial bulk processing. This further expansion is referredto herein as the second expansion, which can include expansion processesgenerally referred to in the art as a rapid expansion process. Thesecond expansion is generally accomplished using a culture mediacomprising a number of components, including feeder cells, a cytokinesource, and an anti-CD3 antibody, in a gas-permeable or gas exchangingcontainer.

In some embodiments, the second expansion or second TIL expansion of TILcan be performed using any TIL culture flasks or containers known bythose of skill in the art. In some embodiments, the second TIL expansioncan proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, or 14 days. In some embodiments, the second TIL expansion canproceed for about 7 days to about 14 days. In some embodiments, thesecond TIL expansion can proceed for about 8 days to about 14 days. Insome embodiments, the second TIL expansion can proceed for about 9 daysto about 14 days. In some embodiments, the second TIL expansion canproceed for about 10 days to about 14 days. In some embodiments, thesecond TIL expansion can proceed for about 11 days to about 14 days. Insome embodiments, the second TIL expansion can proceed for about 12 daysto about 14 days. In some embodiments, the second TIL expansion canproceed for about 13 days to about 14 days. In some embodiments, thesecond TIL expansion can proceed for about 14 days.

In an embodiment, the second expansion can be performed in a gaspermeable container using the methods of the present disclosure. Forexample, TILs can be rapidly expanded using non-specific T-cell receptorstimulation in the presence of interleukin-2 (IL-2) or interleukin-7(IL-7) or interleukin-15 (IL-15); IL-12. The non-specific T-cellreceptor stimulus can include, for example, an anti-CD3 antibody, suchas about 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody(commercially available from Ortho-McNeil, Raritan, N.J. or MiltenyiBiotech, Auburn, Calif) or UHCT-1 (commercially available fromBioLegend, San Diego, Calif, USA). TILs can be expanded to inducefurther stimulation of the TILs in vitro by including one or moreantigens during the second expansion, including antigenic portionsthereof, such as epitope(s), of the cancer, which can be optionallyexpressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217(210M), optionally in the presence of a T-cell growth factor, such as300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g.,NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, andVEGFR2, or antigenic portions thereof. TIL may also be rapidly expandedby re-stimulation with the same antigen(s) of the cancer pulsed ontoHLA-A2-expressing antigen-presenting cells. Alternatively, the TILs canbe further re-stimulated with, e.g., example, irradiated, autologouslymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.In some embodiments, the re-stimulation occurs as part of the secondexpansion. In some embodiments, the second expansion occurs in thepresence of irradiated, autologous lymphocytes or with irradiatedHLA-A2+ allogeneic lymphocytes and IL-2.

In an embodiment, the cell culture medium further comprises IL-2. Insome embodiments, the cell culture medium comprises about 3000 IU/mL ofIL-2. In an embodiment, the cell culture medium comprises about 100IU/mL, about 200 IU/mL, about 300 IU/mL, about 400 IU/mL, about 500IU/mL, about 600 IU/mL, about 700 IU/mL, about 800 IU/mL, about 900IU/mL, 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL,about 3000 IU/mL, about 3500 IU/mL, about 40001U/mL, about 4500 IU/mL,about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL,about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In anembodiment, the cell culture medium comprises between 1000 and 2000IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In an embodiment, the cell culture medium comprises OKT3 antibody. Insome embodiments, the cell culture medium comprises about 30 ng/mL ofOKT3 antibody. In an embodiment, the cell culture medium comprises about0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about ng/mL,about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL,about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL ofOKT3 antibody. In an embodiment, the cell culture medium comprisesbetween 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL,and between 50 ng/mL and 100 ng/mL of OKT3 antibody.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the second expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the second expansion. In someembodiments, a combination of IL-2, IL-15, and IL-21 are employed as acombination during the second expansion. In some embodiments, IL-2,IL-15, and IL-21 as well as any combinations thereof can be included.

In some embodiments, the second expansion can be conducted in asupplemented cell culture medium comprising IL-2, OKT-3, andantigen-presenting feeder cells. In some embodiments, the secondexpansion occurs in a supplemented cell culture medium. In someembodiments, the supplemented cell culture medium comprises IL-2, OKT-3,and antigen-presenting feeder cells. In some embodiments, the secondcell culture medium comprises IL-2, OKT-3, and antigen-presenting cells(APCs; also referred to as antigen-presenting feeder cells). In someembodiments, the second expansion occurs in a cell culture mediumcomprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e.,antigen presenting cells).

In some embodiments, the second expansion culture media comprises about500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the second expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the second expansion culture media comprises about 400IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, thesecond expansion culture media comprises about 300 IU/mL of IL-15 toabout 100 IU/mL of IL-15. In some embodiments, the second expansionculture media comprises about 200 IU/mL of IL-15. In some embodiments,the cell culture medium comprises about 180 1U/mL of IL-15. In anembodiment, the cell culture medium further comprises IL-15. In apreferred embodiment, the cell culture medium comprises about 180 IU/mLof IL-15.

In some embodiments, the second expansion culture media comprises about20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21,about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21,about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21,or about 0.5 IU/mL of IL-21. In some embodiments, the second expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the second expansion culture media comprisesabout 15 IU/mL of IL-21 to about IU/mL of IL-21. In some embodiments,the second expansion culture media comprises about 12 IU/mL of IL-21 toabout 0.5 IU/mL of IL-21. In some embodiments, the second expansionculture media comprises about 10 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the second expansion culture media comprisesabout 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In some embodiments,the second expansion culture media comprises about 2 IU/mL of IL-21. Insome embodiments, the cell culture medium comprises about 1 IU/mL ofIL-21. In some embodiments, the cell culture medium comprises about 0.5IU/mL of IL-21. In an embodiment, the cell culture medium furthercomprises IL-21. In a preferred embodiment, the cell culture mediumcomprises about 1 IU/mL of IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) arePBMCs. In an embodiment, the ratio of TILs to PBMCs and/orantigen-presenting cells in the rapid expansion and/or the secondexpansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225,about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In anembodiment, the ratio of TILs to PBMCs in the rapid expansion and/or thesecond expansion is between 1 to 50 and 1 to 300. In an embodiment, theratio of TILs to PBMCs in the rapid expansion and/or the secondexpansion is between 1 to 100 and 1 to 200.

In an embodiment, REP and/or the second expansion is performed in flaskswith the bulk TILs being mixed with a 100- or 200-fold excess ofinactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mLIL-2 in 150 ml media. Media replacement is done (generally ⅔ mediareplacement via respiration with fresh media) until the cells aretransferred to an alternative growth chamber. Alternative growthchambers include G-REX flasks and gas permeable containers as more fullydiscussed below.

In some embodiments, the second expansion (which can include processesreferred to as the REP process) is shortened to 7-14 days, as discussedin the examples and figures. In some embodiments, the second expansionis shortened to 11 days.

In an embodiment, REP and/or the second expansion may be performed usingT-175 flasks and gas permeable bags as previously described (Tran, etal., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother.2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks). In someembodiments, the second expansion (including expansions referred to asrapid expansions) is performed in T-175 flasks, and about 1×10⁶ TILssuspended in 150 mL of media may be added to each T-175 flask. The TILsmay be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplementedwith 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175flasks may be incubated at 37° C. in 5% CO2. Half the media may beexchanged on day 5 using medium with 3000 IU per mL of IL-2. In someembodiments, on day 7 cells from two T-175 flasks may be combined in a 3L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL ofIL-2 was added to the 300 ml of TIL suspension. The number of cells ineach bag was counted every day or two and fresh media was added to keepthe cell count between 0.5 and 2.0×10⁶ cells/mL.

In an embodiment, the second expansion may be performed in 500 mLcapacity gas permeable flasks with 100 cm gas-permeable silicon bottoms(G-Rex 100, commercially available from Wilson Wolf ManufacturingCorporation, New Brighton, Minn., USA), 5×10⁶ or 10×10⁶ TIL may becultured with PBMCs in 400 mL of 50/50 medium, supplemented with 5%human AB serum, 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3(OKT3). The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2. Onday 5, 250 mL of supernatant may be removed and placed into centrifugebottles and centrifuged at 1500 rpm (491×g) for 10 minutes. The TILpellets may be re-suspended with 150 mL of fresh medium with 5% human ABserum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100flasks. When TIL are expanded serially in G-Rex 100 flasks, on day 7 theTIL in each G-Rex 100 may be suspended in the 300 mL of media present ineach flask and the cell suspension may be divided into 3 100 mL aliquotsthat may be used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask.The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2 and after 4days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to eachG-REX 100 flask. The cells may be harvested on day 14 of culture.

In an embodiment, the second expansion (including expansions referred toas REP) is performed in flasks with the bulk TILs being mixed with a100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media. In someembodiments, media replacement is done until the cells are transferredto an alternative growth chamber. In some embodiments, ⅔ of the media isreplaced by respiration with fresh media. In some embodiments,alternative growth chambers include G-REX flasks and gas permeablecontainers as more fully discussed below.

In an embodiment, the second expansion (including expansions referred toas REP) is performed and further comprises a step wherein TILs areselected for superior tumor reactivity. Any selection method known inthe art may be used. For example, the methods described in U.S. PatentApplication Publication No. 2016/0010058 A1, may be used for selectionof TILs for superior tumor reactivity.

Optionally, a cell viability assay can be performed after the secondexpansion (including expansions referred to as the REP expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. In some embodiments, TILsamples can be counted and viability determined using a Cellometer K2automated cell counter (Nexcelom Bioscience, Lawrence, Mass.).

In some embodiments, the second expansion (including expansions referredto as REP) of TIL can be performed using T-175 flasks and gas-permeablebags as previously described (Tran K Q, Zhou J, Durflinger K H, et al.,2008, J Immunother., 31:742-751, and Dudley M E, Wunderlich J R, SheltonT E, et al. 2003, J Immunother., 26:332-342) or gas-permeable G-Rexflasks. In some embodiments, the second expansion is performed usingflasks. In some embodiments, the second expansion is performed usinggas-permeable G-Rex flasks. In some embodiments, the second expansion isperformed in T-175 flasks, and about 1×10⁶ TIL are suspended in about150 mL of media and this is added to each T-175 flask. The TIL arecultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells at aratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CMand AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at 37° C. in 5%CO2. In some embodiments, half the media is changed on day 5 using 50/50medium with 3000 IU/mL of 1L-2. In some embodiments, on day 7, cellsfrom 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with5% human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TILsuspension. The number of cells in each bag can be counted every day ortwo and fresh media can be added to keep the cell count between about0.5 and about 2.0×10⁶ cells/mL.

In some embodiments, the second expansion (including expansions referredto as REP) are performed in 500 mL capacity flasks with 100 cm²gas-permeable silicon bottoms (G-Rex 100, Wilson Wolf), about 5×10⁶ or10×10⁶ TIL are cultured with irradiated allogeneic PBMC at a ratio of 1to 100 in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2and 30 ng/mL of anti-CD3. The G-Rex 100 flasks are incubated at 37° C.in 5% CO2. In some embodiments, on day 5, 250 mL of supernatant isremoved and placed into centrifuge bottles and centrifuged at 1500 rpm(491 g) for 10 minutes. The TIL pellets can then be resuspended with 150mL of fresh 50/50 medium with 3000 IU/mL of IL-2 and added back to theoriginal G-Rex 100 flasks. In embodiments where TILs are expandedserially in G-Rex 100 flasks, on day 7 the TIL in each G-Rex 100 aresuspended in the 300 mL of media present in each flask and the cellsuspension was divided into three 100 mL aliquots that are used to seed3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000IU/mL of IL-2 is added to each flask. The G-Rex 100 flasks are incubatedat 37° C. in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU/mL ofIL-2 is added to each G-Rex 100 flask. The cells are harvested on day 14of culture.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained in thesecond expansion exhibit an increase in the T-cell repertoire diversity.In some embodiments, the increase in diversity is an increase in theimmunoglobulin diversity and/or the T-cell receptor diversity. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin heavy chain. In some embodiments, the diversity is in theimmunoglobulin is in the immunoglobulin light chain. In someembodiments, the diversity is in the T-cell receptor. In someembodiments, the diversity is in one of the T-cell receptors selectedfrom the group consisting of alpha, beta, gamma, and delta receptors. Insome embodiments, there is an increase in the expression of T-cellreceptor (TCR) alpha and/or beta. In some embodiments, there is anincrease in the expression of T-cell receptor (TCR) alpha. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) beta. In some embodiments, there is an increase in the expressionof TCRab (i.e., TCRαβ).

In some embodiments, the second expansion culture medium (e.g.,sometimes referred to as CM2 or the second cell culture medium),comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells(APCs).

In some embodiments, the second expansion is performed in a closedsystem bioreactor. In some embodiments, a closed system is employed forthe TIL expansion, as described herein. In some embodiments, a singlebioreactor is employed. In some embodiments, the single bioreactoremployed is for example G-REX-10 or a G-REX-100 or advantageously thedevice of WO 2018/130845. In some embodiments, the closed systembioreactor is a single bioreactor.

In an embodiment, the second expansion procedures described herein, aswell as those referred to as REP) require an excess of feeder cellsduring REP TIL expansion and/or during the second expansion. In manyembodiments, the feeder cells are peripheral blood mononuclear cells(PBMCs) obtained from standard whole blood units from healthy blooddonors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the REP procedures, which provides anexemplary protocol for evaluating the replication incompetence ofirradiate allogeneic PBMCs.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells on day 14 is less than the initial viablecell number put into culture on day 0 of the REP and/or day 0 of thesecond expansion (i.e., the start day of the second expansion).

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 30 ng/ml OKT3antibody and 3000 IU/ml IL-2.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TM expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 5-60 ng/ml OKT3antibody and 1000-6000 IU/ml IL-2. In some embodiments, the PBMCs arecultured in the presence of 10-50 ng/ml OKT3 antibody and 2000-5000IU/ml IL-2. In some embodiments, the PBMCs are cultured in the presenceof 20-40 ng/ml OKT3 antibody and 2000-4000 IU/ml IL-2. In someembodiments, the PBMCs are cultured in the presence of 25-ng/ml OKT3antibody and 2500-3500 IU/ml IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. Insome embodiments, the antigen-presenting feeder cells are artificialantigen-presenting feeder cells. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is about 1 to25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375,about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILsto antigen-presenting feeder cells in the second expansion is between 1to 50 and 1 to 300. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is between 1 to100 and 1 to 200.

In an embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10 9 feeder cells to about 100×10⁶ TILs. Inanother embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 50×10⁶ TILs. Inyet another embodiment, the second expansion procedures described hereinrequire about 2.5×10 9 feeder cells to about 25×10⁶ TILs.

In an embodiment, the second expansion procedures described hereinrequire an excess of feeder cells during the second expansion. In manyembodiments, the feeder cells are peripheral blood mononuclear cells(PBMCs) obtained from standard whole blood units from healthy blooddonors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation. In an embodiment, artificialantigen-presenting (aAPC) cells are used in place of PBMCs.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the TIL expansion procedure.

In an embodiment, artificial antigen presenting cells are used in thesecond expansion as a replacement for, or in combination with, PBMCs.

The expansion methods described herein generally use culture media withhigh doses of a cytokine, in particular IL-2, as is known in the art.

Alternatively, using combinations of cytokines for the rapid expansionand or second expansion of TILS is additionally possible, withcombinations of two or more of IL-2, IL-15 and IL-21 as is generallyoutlined in International Publication No. WO 2015/189356 and WInternational Publication No. WO 2015/189357, hereby expresslyincorporated by reference in their entirety. Thus, possible combinationsinclude IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15and IL-21, with the latter finding particular use in many embodiments.The use of combinations of cytokines specifically favors the generationof lymphocytes, and in particular T-cells as described therein.

In some embodiments, the culture media used in expansion methodsdescribed herein (including those referred to as REP) also includes ananti-CD3 antibody. An anti-CD3 antibody in combination with IL-2 inducesT cell activation and cell division in the TIL population. This effectcan be seen with full length antibodies as well as Fab and F(ab′)2fragments, with the former being generally preferred; see, e.g., Tsoukaset al., J. Immunol. 1985, 135, 1719, hereby incorporated by reference inits entirety.

As will be appreciated by those in the art, there are a number ofsuitable anti-human CD3 antibodies that find use in the invention,including anti-human CD3 polyclonal and monoclonal antibodies fromvarious mammals, including, but not limited to, murine, human, primate,rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3antibody is used (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.).

After the second expansion step, cells can be harvested. In someembodiments the TILs are harvested after one, two, three, four or moreexpansion steps. In some embodiments the TILs are harvested after twoexpansion steps.

TILs can be harvested in any appropriate and sterile manner, includingfor example by centrifugation. Methods for TIL harvesting are well knownin the art and any such know methods can be employed with the presentprocess. In some embodiments, TILS are harvested using an automatedsystem.

Cell harvesters and/or cell processing systems are commerciallyavailable from a variety of sources, including, for example, FreseniusKabi, Tomtec Life Science, Perkin Elmer, and Inotech BiosystemsInternational, Inc. Any cell based harvester can be employed with thepresent methods. In some embodiments, the cell harvester and/or cellprocessing systems is a membrane-based cell harvester. In someembodiments, cell harvesting is via a cell processing system, such asthe LOVO system (manufactured by Fresenius Kabi). The term “LOVO cellprocessing system” also refers to any instrument or device manufacturedby any vendor that can pump a solution comprising cells through amembrane or filter such as a spinning membrane or spinning filter in asterile and/or closed system environment, allowing for continuous flowand cell processing to remove supernatant or cell culture media withoutpelletization. In some embodiments, the cell harvester and/or cellprocessing system can perform cell separation, washing, fluid-exchange,concentration, and/or other cell processing steps in a closed, sterilesystem.

In some embodiments, the harvest is performed from a closed systembioreactor. In some embodiments, a closed system is employed for the TILexpansion, as described herein. In some embodiments, a single bioreactoris employed. In some embodiments, the single bioreactor employed is forexample G-REX-10 or a G-REX-100 or advantageously the device of WO2018/130845. In some embodiments, the closed system bioreactor is asingle bioreactor.

Cells are transferred to a container for use in administration to apatient. In some embodiments, once a therapeutically sufficient numberof TILs are obtained using the expansion methods described above, theyare transferred to a container for use in administration to a patient.

In an embodiment, TILs expanded using APCs of the present disclosure areadministered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of TILs in asterile buffer. TILs expanded using PBMCs of the present disclosure maybe administered by any suitable route as known in the art. In someembodiments, the T-cells are administered as a single intra-arterial orintravenous infusion, which preferably lasts approximately 30 to 60minutes. Other suitable routes of administration includeintraperitoneal, intrathecal, and intralymphatic.

In an embodiment, TILs expanded using the methods of the presentdisclosure are administered to a patient as a pharmaceuticalcomposition. In an embodiment, the pharmaceutical composition is asuspension of TILs in a sterile buffer. TILs expanded using PBMCs of thepresent disclosure may be administered by any suitable route as known inthe art. In some embodiments, the T-cells are administered as a singleintra-arterial or intravenous infusion, which preferably lastsapproximately 30 to 60 minutes. Other suitable routes of administrationinclude intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. In some embodiments, fromabout 2.3×10¹⁰ to about 13.7×10¹⁰ TILs are administered, with an averageof around 7.8×10¹⁰ TILs, particularly if the cancer is melanoma. In anembodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs are administered.In some embodiments, about 3×10¹⁰ to about 12×10¹⁰ TILs areadministered. In some embodiments, about 4×10¹⁰ to about 10×10¹⁰ TILsare administered. In some embodiments, about 5×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 6×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 7×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, the therapeutically effectivedosage is about 2.3×10¹⁰ to about 13.7×10¹⁰. In some embodiments, thetherapeutically effective dosage is about 7.8×10¹⁰ TILs, particularly ofthe cancer is melanoma. In some embodiments, the therapeuticallyeffective dosage is about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs. In someembodiments, the therapeutically effective dosage is about 3×10¹⁰ toabout 12×10¹⁰ TILs. In some embodiments, the therapeutically effectivedosage is about 4×10¹⁰ to about 10×10¹⁰ TILs. In some embodiments, thetherapeutically effective dosage is about 5×10¹⁰ to about 8×10¹⁰ TILs.In some embodiments, the therapeutically effective dosage is about6×10¹⁰ to about 8×10¹⁰ TILs. In some embodiments, the therapeuticallyeffective dosage is about 7×10¹⁰ to about 8×10¹⁰ TILs.

In some embodiments, the number of the TILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰ 1×10¹⁰, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹²,5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³,5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the numberof the TILs provided in the pharmaceutical compositions of the inventionis in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, to 1×10¹², 1×10¹² to 5×10¹²,and 5×10¹² to 1×10¹³.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%,0.001%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or0.0001% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is greater than 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%,13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%11%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%,7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%,4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%,2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%,0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.007%, 0.006%,0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0006%,0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of thepharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% toabout 27%, about 0.05% to about 26%, about 0.06% to about 25%, about0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% toabout 16%, about 0.7% to about 15%, about 0.8% to about 14%, about toabout 12% or about 1% to about 10% w/w, w/v or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%,about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% toabout 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/vor v/v of the pharmaceutical composition.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g,0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, g, 0.09 g, 0.08 g, 0.07 g, 0.06 g,0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g,0.0007 g, 0.0006 g, g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, g, 0.0007 g, 0.0008 g, 0.0009 g,0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, g, 0.0045 g,0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g,0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g,0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g,5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.

The TILs provided in the pharmaceutical compositions of the inventionare effective over a wide dosage range. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of theTILs may also be used if appropriate. The amounts of the pharmaceuticalcompositions administered using the methods herein, such as the dosagesof TILs, will be dependent on the human or mammal being treated, theseverity of the disorder or condition, the rate of administration, thedisposition of the active pharmaceutical ingredients and the discretionof the prescribing physician.

In some embodiments, TILs may be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, TILs may be administered in multiple doses. Dosing may beonce, twice, three times, four times, five times, six times, or morethan six times per year. Dosing may be once a month, once every twoweeks, once a week, or once every other day. Administration of TILs maycontinue as long as necessary.

In some embodiments, an effective dosage of TILs is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, an effectivedosage of TILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10 9 to5×10⁹, 5×10 9 to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹¹ to 1×10¹¹, 5×10¹¹ to1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of TILs is in the range ofabout 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of TILs is in the range ofabout 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg toabout 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg,about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg toabout 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg,about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg toabout 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg,or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg,about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the TILs may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, including intranasal and transdermal routes,by infra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, topically, bytransplantation, or by inhalation.

The present invention also includes kits useful in performing diagnosticand prognostic assays using the TILs, in particular UTILs, of thepresent invention. Kits of the invention include buffers, cytokines,flasks, media, product containers, reagents and instructions.

A non-limiting multi-step embodiment is presented below to set up TILgrowth out from a tumor, a setup of a rapid expansion process,confirmation that irradiated PBMC feeders are not expanding and atransfer of static culture to a WAVE bioreactor (see, e.g.,https://www.gelifesciences.com/en/us/shop/cell-culture-and-fermentation/rocking-bioreactors/consumables-and-accessories/single-use-readytoprocess-wave-cellbag-bioreactors-p-00346#overview)and formulation and fill.

In step one (Day 0), the cryopreserved disaggregated tumor tissue isthawed and resuspended 1:9 in T cell culture media supplemented with 10%FBS and 3000 IU/mL IL-2 prior to filtration through an inline 100-270 μmfilter and centrifugation in a 50 mL centrifuge tube prior toresuspension in 20 mL. A sample is taken for flow cytometry analysisSOP-to quantify a number of HLA-A, B, C and CD58⁺, and DRAQ7⁻ cells.

In step two, the cell suspension is then seeded at ≥0.25×10⁶ to≤0.75×10⁶ HLA-A,B,C & CD58⁺ and DRAQ7⁻ cells/mL in CM-T (T cell mediasupplemented with 10% Fetal Bovine Serum) supplemented with addedantibacterial and antifungal agents (Amphotericin B & Gentamicin) andinterleukin-2 (IL-2) 1000 IU/ml in cell culture containers. The T cellsare grown out over 2 week period in CM-T from day 5 half the media isremoved and replaced with fresh media CM-T supplemented with 10% FetalBovine Serum, Amphotericin B & Gentamicin and IL-2. This is repeatedevery ⅔ days between day 5 and day 10 to ensure the cells are maintainedat <0.1×10⁶ to 2×10⁶ CD45⁺ CD3⁺ Annexin-V^(−ve) DRAQ7^(−ve) cells/mL. Amicrobial examination test of TIL culture supernatant (Day 5-7) by PHEur 2.6.27 confirms no microbial growth. Flow cytometry analysis (Day7-10) quantifies a concentration of CD45⁺ CD3⁺ Annexin-V⁻ & DRAQ7⁻cells.

In step three, isolate 4×10⁹ irradiated PBMCs (25 to 50 Gy) with Ficoll(Density 1.078 g/ml) from multiple allogeneic donors (healthy blooddonation derived Buffy coat). Flow cytometry analysis quantifies CD45⁺Annexin-V⁻, and DRAQ7⁻ cells. A microbial examination test of irradiatedPBMCs determines microbial growth.

In step four, the amount of TIL available for the start of the rapidexpansion process is quantified (Day-12). Flow cytometry analysisquantifies CD45⁺ CD3⁺ Annexin-V⁻, and DRAQ7⁻ cells.

In step 5, a culture mixture of feeders (Irradiated ficoll isolatedPBMCs) is prepared and growth supplements in 3L of T cell mixed mediacontaining: ≥3 to <5×10⁹ Irradiated PBMCs—CD45⁺ Annexin-V⁻, and DRAQ7⁻cells, 7-9% human AB serum, 2000 to 4000 IU/mL IL-2 and to 40 ng/mlOKT-3 antibody in a closed static cell culture bag.

In step 6, a representative sample of the culture mixture of feeders(Irradiated ficoll isolated PBMCs) is taken for a control flask prior toadding TIL.

In step 7, TIL is added to a REP culture: ≥1 to <20×10⁶ Tumor derivedTIL—CD45⁺ CD3⁺ Annexin-V⁻, and DRAQ7⁻ cells.

In step 8, static culture is incubated between 35 to 38.5° C. with 3.5to 6% Carbon dioxide in a dry incubator for 6 days. The number andviability of CD45⁺ Annexin-V⁻, and DRAQ7⁻ cells are assessed in theControl flask (collected at Step 6) at Day 14 and 18 containing the REPmixture without TIL to ensure irradiated feeders are not expanding. Flowcytometry analysis quantifies CD45⁺ Annexin-V⁻, and DRAQ7⁻ cells.

In step 9, a WAVE bioreactor bag is preconditioned for 1-2 hours at 35to 38.5° C. with 3.5 to 6% carbon dioxide with 1.7 L of TCM supplementedwith: 7-9% Human AB serum and 2000 to 4000 IU/mL IL-2.

In step 10, TIL is transferred and expanded in the WAVE bioreactorsystem.

In step 11, a perfusion feed 1×TCM 10 L bag supplemented with 2000 to4000 IU/mL IL-2 is connected.

In step 12 (days 19-22), the perfusion rate between day 19 and day 22 isadjusted.

In step 13, (day 24), perfusion is stopped, and waste and feed isdisconnected.

In step 14, TIL is concentrated and washed.

In step 15, a final drug formulation is made with cells suspended in PBScontaining 10% DMSO and 8.5% HSA in a total volume range of 125 to 270mL transfusion bag.

In step 16, a sample of the final product bag containing TIL is takenfor QC assay and retention samples. The QC assays of the fresh drugproduct include microbial examination testing and color and visibleparticle testing. Retention samples are prepared for cell dose,viability phenotype and potency; microbial examination and endotoxinanalysis.

In step 17, the final product container is labeled and overlapped with afinal product label.

In step 18, there is cryopreservation by controlled rate freezing at −1°C./minute to −60° C. and a transfer to ≤−130° C. storage. QC assays forthe cryopreserved drug product include mycoplasma testing by qPCR, Tcell dose and viability testing, endotoxin testing as measured using akinetic chromogenic LAL test and potency testing to assess the CD2⁺Expressing CD45⁺ DRAQ7⁻ for a combination of CD137⁺, IFN-γ⁺, TNFα+, orCD107a⁺ after co-culture with a cell line expressing an anti-CD3fragment.

TABLE 1 Overview manufacture using static culture bags only TumourViable derived TIL CD3+ cells Final Issue from first in second ViableCTU-TIL expansion expansion Fold CD3+ # (Sex) (×10⁷) (×10⁷) Expansion*(×10¹⁰) 1 (F) 2.1 1.5 690 1.0 3 (M) 3.8 2.0 1100 2.2 5 (M) 8.2 1.5 12812.0 Mean ± SD 6.0 ± 2.2 1.7 ± 0.23 1023 ± 303 1.7 ± 0.52 *Equals Finalmanufactured TIL/TIL used in REP

TABLE 2 Overview manufacture using perfusion bioreactor Tumour Viablederived CD3+ cells Final Issue from first in second Viable CTU-TILexpansion expansion Fold CD3+ # (Sex) (×10⁷) (×10⁷) Expansion* (×10¹⁰)12 (F) 5.4 2.0 1600 3.2 13 (M) 14.0 2.0 1010 2.02 14 (M) 5.8 2.0 21004.2 15 (M) 5.1 2.0 3100 6.2 16 (M) 3.0 1.8 3000 5.4 19 (M) 7.6 2.0 34006.8 20 (M) 1.4 1.1 5409 5.95 21 (F) 1.4 1.4 3646 4.85 27 (M) 5.1 2.01845 3.69 28 (F) 8.9 2.0 1590 3.18 32 (F) 34.0 2.0 1835 3.67 32 (F) N/A** 2.0 1985 3.97 35 (M) 8.6 2.0 3125 6.25 36 (M) 3.2 1.6 2050 3.28 37(F) 4.0 2.0 1265 2.53 38 (M) 0.55 0.32 3969 1.27 39 (M) 0.83 0.83 13981.16 40 (F) 1.4 0.71 7444 5.3 41 (M) 9.0 2.0 1555 3.11 42 (M) 9.8 2.01965 3.93 43 (F) 25.0 2.0 2310 4.62 47 (F) 2.67 2.0 1450 2.9 48 (F) 2.732.0 1865 3.73 51 (M) 4.1 2.0 1780 3.56 54 (M) 27.5 2.0 395 7.9 57 (M)2.3 1.5 764 1.13 60 (F) 3.1 1.1 1486 1.56 63 (M) 0.84 0.89 5842 5.24 64(M) 0.72 0.72 2993 2.14 67 (M) 0.38 0.37 7526 2.82 Mean ± SD 6.61 ± 8.21.56 ± 0.61 2650 ± 1770 3.52 ± 1.69 *Equals Final manufactured TIL/TILused in REP ** Patient treated twice using original tumour derived TIL

The present invention provides a disaggregation system or device. Insome embodiments, the disaggregation device is in the form of a treadingdevice for disaggregation of tissue into individual cells or cellclumps. In some embodiments, the disaggregation device provides thermalcontrol during the disaggregation process. In some embodiments, theinvention provides a cryopreservation system or device. In someembodiments, there is provided a device for disaggregation andcryopreservation and thermal control is provided. In another aspect, theinvention provides one or more flexible containers, or a systemcontaining a plurality of containers comprising one or more flexiblecontainers adapted for disaggregation, cryopreservation, or bothdisaggregation and cryopreservation in a disaggregation/cryopreservationsystem or device of the invention. In some embodiments, the one or morecontainers or the plurality of containers are interconnected andsuitable for use in a closed system. The above-mentioned aspects arerepresented in the claims appended herein. More advantages and benefitsof the present invention will become readily apparent to the personskilled in the art in view of the detailed description below whichprovides examples of the invention.

In certain embodiments a disaggregator comprises one or more movablesurfaces, for example plates and/or paddles, and is designed to applycompression and shear forces to a tissue sample. In an embodiment, thedigester comprises a first surface and a second surface that are capableof moving relative to one another. In certain embodiments, the surfacesare opposing surfaces disposed to apply pressure to a sample. In anembodiment, at least one of the surfaces is moved in a directionperpendicular to the direction of the surfaces so as to apply pressureto a sample. In an embodiment, the surfaces are aligned in parallel anddesigned to move together and apart in a repeated or cyclical mannersuch that a sample is repeatedly compressed then relaxed between thesurfaces in a cyclical manner. In embodiments of the invention,compression and relaxation of the sample results in shear forces in thesample.

In an embodiment, one of the first and second surfaces is heldstationary while the other surface is moved. In another embodiment, bothof the first and second surfaces are moved. In an embodiment, the tissuesample is contained in a flexible and/or elastic container whichcontains the tissue sample and optionally disaggregation fluid orsolution. In certain embodiments, the container accommodates changes involume between the first and second surfaces as the surfaces are moved.In certain embodiments, the container is elastic and confines the tissuesample and disaggregation fluid within the extent of the opposingsurfaces. In certain embodiments, the container is flexible andsurrounding air pressure assists confinement of tissue sample anddisaggregation fluid within the extent of the opposing surfaces. Incertain embodiments, the air pressure is ambient pressure. In certainembodiments, air pressure is applied in an enclosing chamber and thepressure is greater than ambient.

In certain embodiments, the disaggregation device comprises two or moresets of opposing surfaces, disposed side-by-side. In some suchembodiments, one surface is common to the sets, for example a singleplate, optionally held stationary, while the second surfaces of each setare located side-by-side and apply pressure against the stationaryplate. The second surfaces may alternately apply pressure in a treadingmotion. In certain such embodiments, a flexible container is employedthat confines the tissue sample and disaggregation fluid within thespace between the stationary surface and the moving surfaces whileallowing the contents of the container to flow back and forth betweenthe moving surfaces. In certain embodiments, the container is adapted tolimit or prevent such back-and-forth movement of the contents. In anembodiment, a seal across the container blocks flow of contents from oneside to the other. In another embodiment, a baffle across the containerlimits flow of contents from one side to the other.

The treading surfaces can be actuated by any suitable mechanism.Disclosed herein as device 100 is an example of a lateral bar systemdesigned to move treading surfaces alternately against a flexiblecontainer. The treading surfaces are sprung, the springs designed topress the treading surfaces against a container while allowing forvariation in container thickness and particle size variation in thecontainer. In certain embodiments, the springs are preloaded. Alsodisclosed herein as device 200 is an example of a cam actuated designthat features two treading surfaces. In device 200, preloaded springspress treading surfaces against a flexible container and the cammechanism cyclically raises one treading surface, then the other, awayfrom the flexible container. In another embodiment, one or more rockerarms or levers is employed to lift treading surfaces away from thecontainer. In yet another embodiment, the treading surfaces are raisedand lowered hydraulically. In yet another embodiment, the treadingsurfaces are raised and lowered pneumatically. While in the 200 device,there are two treading surfaces alternately contacting thedisaggregation container, in certain embodiments, the actuatingmechanism allows all of the moving surfaces to apply pressuresimultaneously including when the system is at rest. Such a feature isuseful to empty the contents of the disaggregation container at the endof disaggregation process. For example, instead of treading surfacesbeing located at intermediate positions or one raised and one lowered,all of the treading surfaces are lowered against the disaggregationcontainer, squeezing out its contents through attached tubing,optionally filtered, into a secondary receiving container, for example acryopreservation container.

In a fully closed disaggregation and cryopreservation system exemplifiedherein, there is featured automated disaggregation followed by manualfiltration and transfer by a sealed system of syringes and tubes to acryopreservation container and automated cryopreservation.Advantageously, while disaggregated tumor tissue is manually transferredfrom a disaggregation container to a cryopreservation container, thedisaggregation and cryopreservation steps are performed by the sameautomated device programmed to sequentially manage both steps. In otherembodiments, the disaggregation procedure is designed such that attermination, the disaggregated tumor tissues is automatically moved froma disaggregation container to a cryopreservation container. In certainembodiments, a peristaltic pump and valves that contact the connectingtubes control flow of the contents. In certain embodiments, the treadingsurfaces of the disaggregator are disposed to push or squeeze thedisaggregated tumor solution out of the disaggregation container,optionally through a filter, into a cryopreservation container, valvescontrolling flow of the contents. In such embodiments, disaggregationand cryopreservation along with any transfer of material in the closedsystem, are preferably controlled and performed by the same device asexemplified herein.

Several disaggregation systems have been tested and optimized withrespect to variables including force, digestion time, and speed (RPM orcycles per minute). Results and projections using several tissue typeswere determined for combinations of force, time, and speed variablesincluding forces up to and above 60 N, digestion times up to and above60 min, and speeds up to and above 240 RPM. In certain embodiments ofthe invention, the force is from 20-200 N, or 30-120 N, or 30-90 N, or40-60 N, or 10-20 N or 20-30 N, or 30-40 N, or 40-50 N, or N, or 45-50N, or 50-55 N, or 55-60 N, or 60-65 N, or 65-70 N, or 70-75 N, or 75-80N. Typical treading feet have surfaces areas from about 20 to 50 cm².Based on a 30 cm² treading surface, the treading pressure is from0.5-6.5 N/cm², or 1-4 N/cm², or 1-3 N/cm², or 1-2 N/cm², or 1.5-2.5N/cm², or 2-3 N/cm², or 2.5-3.5 N/cm², or 1.5 N/cm^(2±0.5) N/cm², or 2N/cm^(2±0.5) N/cm², or 2.5 N/cm^(2±0.5) N/cm², or 3 N/cm^(2±0.5) N/cm²,or 4 N/cm^(2+0.5) N/cm², or 5 N/cm^(2±0.5) N/cm². Nominal pressure canbe measured using a pressure sensor, preferably correcting for thethickness of a disaggregation container. In certain embodiments, thedisaggregation device incorporates a pressure sensor. In certainembodiments of the invention, the digestion time is 90 min. or less, or75 min. or less, or 60 min. or less, or 50 min. or less, or 5-120 min,or 15-100 min., or 30-90 min., or 40-60 min., or 5-10 min., or 10-20min., or 20-30 min., or min., or 40-45 min. or 45-50 min., or 50-60min., or 60-65 min., or 65-70 min., or 40 min.±5 min. or 45 min.±5 min.,or 50 min.±5 min., or 55 min.±5 min., or 60 min.±5 min., or 65 min.±5min., or 70 min.±5 min. In certain embodiments, the disaggregationdevice operates at from 60-360 RPM. or 120-340 RPM, or 180-300 RPM, or210-270 RPM, 80-160 RPM, or 120-200 RPM, or 160-240 RPM, or 200-280 RPM,or 240-320 RPM, or 280-360 RPM, or 60 RPM±RPM, or 80 RPM±20 RPM, or 100RPM±20 RPM, or 120 RPM±20 RPM, or 140 RPM±RPM, or 160 RPM±20 RPM, or 180RPM±20 RPM, or 200 RPM±20 RPM, or 220 RPM±RPM, or 240 RPM±20 RPM, or 260RPM±20 RPM, or 280 RPM±20 RPM, or 300 RPM±RPM, or 320 RPM±20 RPM, or 340RPM±20 RPM, or 360 RPM±20 RPM.

In certain embodiments, physical disaggregation is continuous. Incertain embodiments, physical disaggregation is periodic or episodic.For example, when a temperature increase is observed in a disaggregationsample, it may be advantageous to briefly slow or halt physicaldisaggregation to reduce or prevent temperature increase or allow thetemperature to equilibrate to a set point. Without being bound bytheory, a temperature increase may occur through physical manipulationof a sample by a disaggregation device, heat transfer from an activetreading mechanism of a device, reduced physical contact or heattransfer from sample to a refrigeration unit while the disaggregationprocess is active, or other reason. In certain embodiments, periodic orepisodic disaggregation may be beneficial to the disaggregation device.In a cam driven device as disclosed herein, life expectancy of the cammechanism may be improved by periodically reversing the direction of camrotation from time to time, thus extending the life of the cam bydistributing wear over both sides of the cam. In embodiments of theinvention, activity periods of physical disaggregation include withoutlimitation, 15-30 sec., 20-sec., 30-60 sec., 45-75 sec., 60-90 sec., atleast 20 sec., at least 30 sec., at least 40 sec, at least 1 min. atleast 1.5 min., or at least 2 min. Durations of inactivity can be,without limitation, 1-10 sec, 10-20 sec., 20-30 sec., 30-40 sec. 40-60sec., 5 sec., 10 sec., 20 sec., 30 sec., 40 sec., 60 sec., sec. 120 sec.or durations in between. The duration of inactivity may be as short asis necessary for the disaggregation device to reverse direction.

In some embodiments, the surfaces are opposing surfaces disposed to movelaterally with respect to one another. In certain such embodiments, thelateral motion comprises linear lateral motion. In certain suchembodiments, the lateral motion comprises orbital lateral motion. Incertain embodiment, there is both linear and orbital lateral motion.

In an embodiment, the opposing surfaces are flat. In an embodiment, atleast one of the surfaces comprises a convex region and disposed to bemoved in a rocking motion against the other surface. One aspect of aconvex surface and rocking motion is to provide a peristalsis-likeaction.

According to the invention, the movement of the surfaces is controlled,such control comprising control of one or more aspect of surfacemovement, including but not limited to velocity, sample compression,system pressure, duration, and cycle frequency. In certain embodiments,one or more aspects of plate movement is constant. In certainembodiments, one or more aspect of plate movement depends on the stateof disaggregation. In certain embodiments, the state of disaggregationis defined by the time of the disaggregation procedure, such as forexample one or more predefined stages such as early, middle, late, ormore precise time periods measured in hours, minutes and seconds. Incertain embodiments, the state of disaggregation is defined by the sizedistribution of tumor pieces. For example, in an embodiment of theinvention, pressure is increased as the size of tumor pieces is reduced.

Examples of Disaggregation Devices and Alternatives

Referring to FIG. 41 there is shown a treading device 100 for thedisaggregation of tissue into individual cells or cell clumps within aclosed and at least initially aseptic generally flat-sided andrelatively thin sample container bag 10. The device includes a housing110 formed from an assembly of parts that can be removably inserted intoa temperature controlled device such as a controlled temperature ratechange freezer, thawer or warmer, for example a commercially availablefreezer known as Via Freeze™, or any other device which provides acontrolled rate change in temperature, shown schematically in FIG. 41and described herein generally as freezer 40. In practice the housingwill include a cover, which is not illustrated. In use the device andbag provide a closed system, to disaggregate tissue e.g. excisedtumours, parts of excised tumours or needle biopsies etc., and to thencryopreserve the resulting cell suspension for subsequent analysiswithout the need to transfer the disaggregated sample out of the bag 10.

The housing 110 has a chassis 112 to which is attached a motor unit 114which includes an electric motor and gearbox, which has an output speedof 10-300 rpm. The output shaft of the motor and gearbox 114 has a crank116 which drives a connecting rod 118, which in turn is pivotablyconnected to a treading mechanism 120, which will be moved through onetreading cycle for each revolution of crank 116, i.e. a treading cyclebetween 0.2 and 6 seconds. In more detail this treading mechanism has aparallelogram four bar linkage, which includes two spaced pivots 122 and124 rigidly mounted to the chassis 112 which pivotably mount two opposedparallel horizontal bars 126 and 128 respectively. Each of thehorizontal bars has two parallel treading bars 130 and 132, pivotablyconnected thereto one on each side of the pivots 122 and 124, togetherforming the parallelogram linkage. The connecting rod 118 isconveniently pivotably held to an extension of the top horizontal bar,such that moving of that extension causes cyclic up and down motion (inthe orientation shown) of the treading bars 130 and 132. To eachtreading bar 130 and 132 is connected a foot assembly 134 and 136 which,by virtue of the above-mentioned cyclic motion, will move up and downwith motion of the crank 116, in a sequentially manner, i.e. when onefoot is up the other will be down and vice versa.

The foot assemblies 134 and 136 each include a flat faced sole plate 138and 140 each plate being spring-mounted to a upper foot frame 142 and144 respectively, by coiled metal springs 146. In the arrangementdescribed above, or an equivalent arrangement if used, the springs 146are preloaded-. In this case the combined preload is preferably 40-80N,more preferably 30-70 N for each foot preferably about 60N. The combinedspring rate is 1-5 N per mm of travel, preferably about 3N per mm, andthe intended foot travel is about 8-12 mm, preferably about 10 mm. Inaddition the surface area of each foot is intended to be about 20 to 50cm², preferably about 35 cm². This results in a notional pressure on thebag of between zero (when the foot lifts off the bag or hassubstantially no load, and up to about 6 N/cm² (about 9 psi). Thepreferred notional pressure is about 2N/cm² (about 3 psi). However,given that the bag may not, at least at the start of the treadingprocess, contain a homogeneous material, then there will be lumps ofmaterial where the force exerted will be concentrated, and so thepressure is described as ‘notional’ which is the idealised situation,for example to provide a minimum pressure resistance of the bag 10exerted toward the end of the treading process.

At the bottom of the chassis is a receiving area 148 for the flexiblebag 10 and adjacent the receiving area 148 is heat transfer plate 150.The area 148 is large enough to admit the sample processing bag 10slidable onto the plate 150 via the front of the chassis (the frontbeing shown in FIG. 41 ). The plate includes an upper surface 151 onwhich the bag 10 sits, and a lower surface 152 which in use is exposedfor externally influenced heating or cooling. The upper surface 151 isgenerally parallel to the sole plates 138 and 140 of each foot, so thatthe sole plates move generally parallel to the surface 151. Put anotherway, the flat sole plates move in a generally perpendicular direction tothe surface 151, which prevents significant side forces on the mechanism120. The plate 150 is formed from metal, preferably aluminium or copperor gold or silver, or alloys containing those metals. Heat conductanceis preferably above 100 and more preferably above 200 W/m K measured at20 degrees Celsius. The thickness of the plate 150 material is about 3mm or less and provides low thermal mass and thus a quicker reaction ofthe contents of the bag 10 to follow temperature changes on the oppositeside of the plate.

With reference additionally to FIGS. 42 and 43 , the device is operatedby supplying electrical current to the motor unit 114, to drive thecrank 116, in this example clockwise as shown by arrows C. The crankcauses the connecting rod 118 to operate the above described treadingmechanism 120. It will be noted that the top and bottom of the stroke ofthe crank, where maximum force is applied to the mechanism 120 coincideswith the lowermost position of each foot assembly 134 and 136. The footassemblies move up and down in the direction of arrows U and D tomassage the sample bag 10 sequentially, such that the contents of thebag 10 have an opportunity to move to one side away from the respectivetreading foot. Since the potentially solid tissue samples in the bag canmove away from the treading foot, and because the sole plates 138 and140 of each foot are spring loaded, with additional resilient travelbeing afforded to the feet even when they are at the bottom of theirstroke, then there is less chance that the mechanism will jam whenlarger tissue masses are intended to be disaggregated. The sequentialtreading action also reduces the chances of the bag 10 rupturing.

FIG. 44 is a plan view of the device 100 described above, but no bag 10is in place in this view. In particular, the relative side-by-sidepositions of the foot assemblies 134 and 136 can be seen, which arespaced and have a collective area viewed in plan, which area is aboutequal the area of the bag 10 when laid flat, but a difference in areasof about plus or minus 10% of the area of the bag 10 has utility.

FIG. 45 shows another plan view of a device 100′ which is similar inconstruction to the device 100 described above, but in this alternativethe motor 113 of the motor unit 114 is arranged transversely to theoutput shaft of its gearbox 115 by the use of a 90 degree gearbox 115,so that the motor 113 does not protrude beyond a backwall 111 of thedevice 100′. Thus, this device 100′ can fit into a smaller freezervolume if needed.

During the above-mentioned disaggregation processing, the forces exertedby the foot assemblies 134 and 136 are reacted by the heat transferplate 150. This means that the sample bag is pressed against the contactsurface 151 of the plate 150 during processing, providing good surfacecontact between the sample bag 10 and the plate's surface 151, andconsequently improved heat energy transfer.

FIGS. 46, 47 and 48 show different embodiments of the flexible samplebag 10 mentioned above. The bag in use is slid into place in thereceiving area 148 in the device 100 or 100′ and sits under the two feet134 and 136 mentioned. Thus, the bag has a generally flat construction,of about up to 12 mm thickness, with some additional compliance in orderto fit tissue samples therein. As can be seen from FIG. 46 oneconstruction of a bag 10 is shown formed from two layers of plasticmaterial sealed only at their periphery 14 to form a central cavity 12,and ports 16 for access into the cavity 12. The bag may be formed fromEVA. In use it is preferred that the ports 16, or at least one of them,is/are large enough, i.e. about 10 mm in diameter or larger, to accept asample which if necessary has been chopped into small pieces and passedinto the bag cavity 12 by means of a syringe. However, it is alsopossible to include a so called ‘zip-lock’ access at the end of the bagopposite the ports, such that large tissue samples can be put into thebag and the bag is then re-sealed. The ‘zip-lock’ can be folded over oneor more times to make a seam, held folded inside a resilient channel orby means of another clamp or clamps (not shown) to reduce the chance ofleakage. The bag 10 can, as an alternative, be opened and tissue can beadded. The bag can then be heat sealed with its contents in place. Thebag 10 includes corner apertures 18 for locating the bag in the devicein use and holding it in place during treading. Whilst the drawings showa bag 10 with one cavity 12, it would be possible to provide a baghaving more than one cavity, for example, two, three, four or fivecavities, for example each of the plural cavities being elongate andhaving an initially open, heat sealable end, and a sealable port at itsother end for the introduction of reagents such as a disaggregationenzyme, and for withdrawing the disaggregated sample once thedisaggregation is complete or substantially complete.

FIG. 47 shows the bag 10 of FIG. 46 mounted in a locating frame 20 bymeans of pegs 24 on the frame which fit into the corner apertures 18.The frame 20 is an alternative way of locating and holding the bag 10 inplace within the device 100/100′. The frame 20 includes location holes22 which cooperate with the device for locating and holding the bag inplace during treading. The frame has an inner open window 26 with asmooth rounded inner edge 23, to accommodate the cavity 12 and treadingfeet 134 and 136 in use. The frame 20 makes loading and unloading of thebag 10 into and out of the device 100/100′ easier.

FIG. 48 shows an alternative frame 20′ which has two generallysymmetrical halves each similar to construction of frame 20. Each framehalf has additionally a flexible shell 30 moulded to the frame 20′, suchthat the two halves come together like a clam shell enveloping the bag10. The top and bottom flexible shells act as a bund if the bag 10inside ruptures in use. This feature is particularly useful forinfectious tissue samples.

Yet another alternative, not shown, a simple bag-in-bag arrangementcould be employed to contain leaks. In yet another alternative, the bagmay include a base which has resilient (at least at room temperature)separate wells, such that aliquots of sample can be removed withoutusing the whole sample, for example after freezing as described below.Alternatively, a sealable bag may be further heat sealed into portionsfor allowing the separation of the sample.

The processing of a sample put into the bag 10 can in one examplelargely follow the steps described in WO2018/130845. In this arrangementthe sealed bag TO containing tissue is suspended in an aqueous solutionwhich may contain digestive enzymes such as collagenases and proteasesto accelerate the breakdown of the tissue, introduced into the bag via aport 16. The bag is here placed on the plate 150 and warmed from, forexample, an external heat source to approximately 35° G to acceleratethe rate of tissue digestion. One important difference proposed here isthat a single sample processing bag is employed, and digestive enzymescan be introduced through one of the ports 16 in the bag prior to orduring disaggregation. The heat transfer plate 150 can be used tointroduce heat energy into the bag by heating the plate on its undersideto provide the desired temperature in the bag for enzymatic action. Thatheat could conveniently come from an electrically heated warming plate,or electric heating elements in or on the plate 150. The amount ofdisaggregation action will depend on numerous parameters, for examplethe size, density and elasticity of the initial tissue sample, and sothe time for disaggregation and the rate of treading will varysignificantly. Too long or overly vigorous treading could lead todecreased cell viability. Thus, the motor unit speed and thedisaggregation period is important. One option to address this problemis to time the processing according to a look-up table which includestimes and output speeds required to disaggregate similar samples.Another option is to measure the instantaneous electrical power orelectrical energy over time needed to perform the disaggregationprocessing, or to measure the force or stress exerted on the pate 150 oranother part of the mechanism, and to stop after a predeterminedthreshold has been reached, to indicate that the sample has beensufficiently disaggregated. As the power/forces/stresses reduce thedisaggregation is closer to completion. Another option is to measurelight absorbance through the bag—the greater the absorbance, the closerthe sample is to complete disaggregation. Once disaggregation iscomplete the bag contents can be transferred, and the cells or otherconstituents of interest can be separated and put back into a fresh bagfor freezing in the device 100/100′. Alternatively, and preferably thewhole disaggregated materials can be left in the bag and device forfreezing. A cryoprotectant is introduced in to the bag through a port16.

Another difference between the present methodology and that described inWO2018/130845 is that once a cryoprotectant is introduced, the devicewith the disaggregated sample and cryoprotectant in the bag is mounted(or remains in) the device, and the whole device is mounted in thefreezer 40 as described above. The base of the freezer is cold and sodraws heat energy from the hag 10 via the heat transfer plate 150. Tocontrol the formation of ice and prevent supercooling of the samplewhile the bag it is being cooled, it can be massaged by the feet 134 and136, in the manner described above, albeit at a slower rate than fordisaggregation, to control ice nucleation and so increase the viabilityof the cells after thawing. Electrical energy can be supplied to themotor unit 114 via a wire conductor to maintain motion of the mechanism120 inside the freezer, e.g. freezer 40 (FIG. 41 ).

Since the device is removeable from the freezer, cleaning after use ismade easier.

When required for use, the frozen disaggregated samples in a bag 10 canbe thawed rapidly in the device 100/100′ by further external heating ofthe plate 150, and/or by partially immersing the device 100/100′ in awarmed water bath, maintained at about 37° C., and the cryoprotectantremoved. In each case the bag can be massaged during thawing. If theenzymes are still present, they too can be removed if needed, forexample by means of filtering. Generally, they will have had little orno effect on the cells during cryopreservation because their action ishalted at low temperatures. All the process manipulations, warming,disaggregation, cooling, freezing and then thawing occur with the samplein the same sealed flexible bag 10, and may be performed in a singledevice. This is not only time and space efficient, but it enables asingle record to capture everything that happened to the sample duringprocessing, e.g. temperatures, durations, disaggregation speed, freezingprotocol, and lessens the chance for errors, such as a sample spendingtoo much time in an uncontrolled environment between processingmachines.

More specific examples of the apparatus and techniques used in tissuesample processing and freezing are given below.

FIG. 49 shows an example of a bag 10 formed from a thermoplasticmaterial such as EVA or PVG film and having an opening 11 for acceptingthe tissue sample T. The bag includes tubing 13 attached to the one ormore ports 16 (FIG. 46 ) which tubing includes one or more branches 17,compression valves 19, and standard Luer-type connectors 15. The singletubing line shown is merely illustrative—the bag 10 may includeadditional parallel tubing connected via plural ports 16.

Once the tissue T is inside the bag 10. the opening 11 can be sealed bya mechanical clamping seal 9, shown closed and sealed in FIG. 50 , andshown open in chain dotted lines in the same Figure, and/or by means ofheat sealing using a heat sealing machine 50 as shown in FIG. 51 a , toproduce a heat-sealed closure strip or strips (for example pluralparallel strips) 8, each method forming the sealed cavity 12 (FIGS. 46,47 and 49 ).

An alternative or additional means for sealing a bag 10 is shown inFIGS. 51 b and 51 c . As shown in FIG. 51 c , the bag 10 after heatsealing at seal 8 can be clamped in a two piece clamp which comprises atop bar 62 and a bottom bar 64 forced together by a pair of screws 66.FIG. 51 b shows the clamp 60 in an exploded condition, but in use thescrews 66 need not be completely removed from the remaining clamp priorto insertion of the bag 10. The top bar 62 has a tapering recess 68, inwhich sits a complementary wedge shaped formation 61 when clamped. Therecess and wedge concentrate the clamping forces at the apex of thewedge 65, providing higher clamping forces at the apex than could beachieved by flat clamping faces. For even more clamping force, the apex65 has a small channel 67 at its peak, which is met in use by acomplementary ridged formation 69, in the top bar. In certainembodiments, the forces are sufficient to negate the need for the heatseal 8. In certain embodiments, the heat seal or other bag sealingmechanism is desired, for example to provide for handling of asample-containing bag outside of the disaggregator. In certainembodiments, the clamping device ensures the integrity of the seal. Theclamping force is further enhanced by the thickness and stiffness of thetop and bottom bar which do not readily bend, and so maintain theclamping force exerted by the screws 66. FIG. 51 c shows the clamp 60 ina clamped condition. Protrusions 63 meet with features of the treadingdevice 100/100′ or 200 (as described below) to inhibit movement of theclamp, and consequently the clamped bag 10 during treading. The outerperiphery and height of the clamp 60 is of a sized and shape to fit in acomplementary part of the sample receiving area 148 (or 248 FIG. 62 etseq), and so afford further location of the clamped bag 10 duringtreading. Although not illustrated, the clamp 60 may incorporate also anadditional frame 20, 20′ as shown in FIGS. 47 and 48 , and such that theclamp is rigidly mounted to one end of the frame and the port(s) 16(FIGS. 46 and 49 ) are supported at the other end of the frame.

With reference to FIG. 52 , in use, once sealed, a digestive enzyme Ecan be introduced into the cavity 12 via the tubing 13, for example byinjecting the enzyme into the bag using a syringe 5 attached to thebranch connection 17. By holding the bag in an upright orientation, aircan then be removed from the cavity 12 by withdrawing the piston of thesyringe 5 as shown in FIG. 53 . Initial mixing of the enzyme E andtissue T can be made by hand as shown in FIG. 54 .

Loading of the bag 10 into the treading device 100 for disaggregationcan then be commenced, either with or without the frame 20/20′ andbunding cover 30, as illustrated in FIG. 55 .

The disaggregation process then takes place as described above. Oncecomplete, which may take between several minutes and several hours forexample around 10 minutes to 7 hours, preferably 40 minutes to 1 hour,the disaggregated liquified sample may be subdivided in to aliquots, forexample using the bag set described above, and an additional samplealiquot bag 7, as shown in FIG. 56 , connected to the branch 17. In thatinstance a syringe 5 is used to draw the liquified sample out of the bag10 in the direction of arrows F, valves 19 a and 19 b are open and valve19 c adjacent the sample aliquot bag 7 is closed. Once sufficient samplehas been withdrawn into the syringe 5, valve 19 b is closed, valve 19 aremains open, and valve 19 c is opened. The syringe is then used toforce the liquids in the direction of arrow F in FIG. 57 , into thesample aliquot bag 7. The tubing 13 of aliquot bag 7 can be heat sealedby means of a clamp heat seal machine 55 and shown in FIG. 58 . Thatprocess can be repeated until sufficient aliquots are obtained or untilthe is no more sample left Bag may be partially divided already to makesealing off each compartment simpler.

As described above, the sample bag 10, can remain in the treading device100 (FIG. and the treading device can then be loaded into a controlledrate temperature change device, in this case the freezer 40 as shown inFIG. 59 . That technique allows treading to continue during freezing, toinhibit ice crystals forming, although in practice the bag 10 can beremoved before freezing, and the freezer 40 then acts only to cool thesample through the heat transfer plate during treading. In thealterative, the aliquot sample bags 7 can take the place of the wholesample bag 10. In another alternative, the freezer 40 can be used togently cool the unprocessed or processed sample to around 4 degreesCelsius by mounting the treading device 100 on top of the freezer 40with its lid open so the base 150 is cooled, as shown in FIG. 60 . Inanother alternative it is possible to remove the base 150 and put thatinto the freezer, with the freezer lid in place, as shown in FIG. 61 .In yet another alternative, not shown, the bags 10, or 7 can be frozendirectly in the freezer 40.

The invention is not to be seen as limited by the embodiments describedabove, but can be varied within the scope of the appended claims as isreadily apparent to the person skilled in the art. For instance, thetreading mechanism described above is preferred because it provideswholly pivoting mechanical interconnections which are less likely to jamin cold conditions than sliding surfaces, but that mechanism could bereplaced with any mechanically equivalent means for treading two or morefeet sequentially. The flat feet described may be replaced with rollerfeet, where the treading motion is from side to side rather than up anddown. The treading described, or its mechanical equivalent, ispreferably at a rate of 2 or 3 treads for each foot per second tooptimise disaggregation and maximise cell recovery, and is a steadytreading, but the treading could be quicker or slower, or intermittent,for different cell types.

Since the device 100/100′ is intended to be placed in a freezer andsubjected to extremely low temperatures (e.g. minus 80 degrees Celsiusor lower), the use of metal parts, particularly those parts like springs146 is preferred since polymeric parts become much more rigid at lowtemperatures. Also, tightly fitting parts, like pistons and cylinders,can become jammed or ill-fitting at very low temperatures so simplepivotable linkages like the mechanism 120 described are preferred.

FIGS. 62, 63 and 64 show an alternative treading device 200, which issimilar in size and function to the device 100 described above. Thedevice 200 has certain differences which are described in more detailbelow.

Referring to FIG. 62 , the principal difference between the device 100and the device 200 is that the device 200 has a treading mechanism 220which is different to the mechanism 120 of device 100. Two treading feet234, 236 driven in a cyclic alternate treading motion, similar to themotion shown in FIGS. 42 and 43 , by a 24 volt DC electric motor 213(FIG. 63 ) which is part of an electric motor unit 214 which has arotary encoder providing feedback to a controller 221 (FIG. 63 ) formonitoring and controlling the speed of the treading motion. The motordrives a cam shaft 224 via a toothed belt 222. The cam shaft includes apair of cams 230, 232 offset at 180 degrees, in this instance, eachprofiled with a cycloidal shape to provide simple harmonic motion of thecam follower. Each cam is operable to move a cam follower assemblyincluding an associated elastomeric follower wheel 225, 227 which ridesover the cam's profile, a follower wheel axle 221, 223 in forcetransmitting relationship with a sprung follower carriage 226, 228. Eachcarriage 226,228 slides in a linear guide 229, and a respective foot234, 236 is connected to the carriage. Each assembly is forced upwardsin turn by a respective one of the follower wheels as it rides the camprofile away from a treading condition together with the foot, as therespective cam is rotated by the motor against the urging force of areturn spring 231. As the cam is rotated further, and the cam profilerecedes, the spring 231 associated with each follower assembly forcesthe assembly and foot downwards with a treading force.

Thereby, the treading force is limited to the spring rate of theassociated follower assembly spring 231 and not the power of the drivemotor. 1. The force applied to the hag is, in use, limited by thesprings because the mechanism drives the feet up and the springs pushthem back down. This makes sure that:

-   -   a. the motor cannot stall (regardless of tumour size or        texture);    -   b. the sample is not compressed with excessive force and the bag        will not split;    -   c. the maximum pressure applied to the bag is lower than the        pressure tested during bag manufacture; and    -   d. As described below, a hinged bag receiving area 248 can        accept a sample bag and any clamp used, without necessarily        pre-positioning the feet. In other words, the feet can be in any        position when accepting a bag, because the hinged sample area        248 is closed against the feet, and if needed any sample can at        that time be compressed by the feet as the hinged area is closed        against the feet.

Referring also to FIGS. 63 and 64 , the device 200 further includes aflexible sealing membrane 241 extending from a device housing 210 to theupper parts of the two feet 234, 236 which provides a fluid resistantand dust seal between the soles of the feet and the remaining parts ofthe treading mechanism 220. That arrangement inhibits mechanismcontamination, should the compressed bag split in service. Whilst amembrane 241 is preferred, the feet could slide in seals, such as lippedseals mounted to a partition dividing the mechanism 220 from the bagarea 248, and achieving similar inhibition of contamination of themechanism should that be needed.

The device 200 further includes heat transfer plate 250, which performsthe same function as the heat transfer plate 150. This plate 250,however, is hinged to one side of the housing at hinge 255 (FIG. 64 ),so that insertion and removal of the bag to be trodden (as shown inFIGS. 46, 47 and 48 ) is easier. The heat transfer plate 250 includes atemperature sensor 256 which allows the temperature of the plate 250 andthe bag receiving area 248 to be monitored and recorded by thecontroller, for quality control. The plate 250 has first and secondsurfaces 251 and 252 with the same function as the surfaces 151 and 152described above.

Each foot is adjustable in height relative to a heat transfer plate 250of the device 200 and an indication of its movement is monitored also bythe controller. Thus, even though the rotary encoder may indicate thatthe motor is turning, a mechanical failure, such as a failure of thetoothed belt 222, may still be detected by the controller, and asuitable action can be implemented, such as raising an alarm.

The device 200 has the same external dimensions as the device 100, andthe device's housing 210 is intended to slide inside the controlled ratefreezer 40 with the freezer lid in place as described above andillustrated in FIG. 61 .

For convenience, terms such as upper, lower, up and down, and moredescriptive terms such as feet, tread and treading have been used todescribe the invention shown in the drawings, but in practice, thedevice shown could be oriented in any manner such that those termsbecome for example inverted or less descriptive in that new orientation.Therefore, no limitation as to orientation should be construed by suchterms or equivalent terms.

The invention provides s device (100/100′) for the disaggregation oftissue samples into individual cells or cell clumps in a closed flexiblebag (10), the device including a mechanical disaggregation mechanism(120) and a tissue sample bag receiving area (148), said device furtherincluding a heat transfer plate (150) for transferring heat energy to orfrom the area (148), the plate having a first plate surface (151)adjacent the area (148) and an opposing surface (152) exposed toexternal thermal influence which faces away from the area (148).

Cryopreservation of the tumor tissue at the time of collection resultedin the ability to separate manufacturing from tumor collection. Thismeans UTIL manufacturing can be planned and performed as a singlemanufacturing process from thaw of the tumor digest through to final TILharvest wash, drug product formulation, filling, labelling andcryopreservation.

Cryopreservation of the final product enabled all release testing to beperformed prior to conditioning chemotherapy and patient treatment to bedislocated from final product manufacture.

Flow cytometry was used to characterize and quantify the manufacturedproducts. TILd are defined as T cells that express the cell surfacemarker CD3 that have been culture derived from a metastatic Tumors bypathology assessment of a representative sample of the startingmaterial. Viability is based on the percentage of all CD3⁺ cells whichdo not bind the early cell death marker Annexin-V and/or the viabilitydye DRAQ7 (equivalent to Trypan blue or PI). Purity is defined as thepercentage of viable T cells (CD3⁺, Annexin-V-^(ve), and DRAQ7-^(ve))within the Viable Hematopoietic cell population (CD45⁺, Annexin-V-^(ve),and DRAQ7-^(ve)).

The vast majority of cells prior to the rapid expansion protocol (REP)are T cells expressing CD3. In research as well as clinical batches avariable distribution of CD3+CD8+ and CD3+CD4+ TIL are observed andthese will comprise of a subset containing the tumor-reactive cells. Asthe TTLs are expanded in the REP with anti-CD3, the final productcontains almost exclusively viable CD3+ T cells (>94%).

Theoretically, the end product could still contain tumor cells althoughthis is very unlikely due to the culture conditions that strongly andselectively promote T cell growth and T cell-mediated killing of tumorcells. Clinical data of several hundred TIL infusions have shown nopresence of tumor cells by cytology. In order to collate data toultimately set a specification, a test has been incorporated to identifyall viable cellular material that is not hematopoietic in origin IPCassay and will also test for a frequency of cancer biomarkers.

A TIL cell drug product is a suspension in approximately 125-270 ml ofbuffered isotonic saline containing 8.5% Human Serum Albumin and 10%DMSO. The number of cells present is dependent on the ability of eachindividual's TIL cells to be expanded in culture in conjunction with theculture conditions and the manufacturing reproducibility.

TABLE 3 Exemplary Drug Product Composition Quantity Component (perinfusion bag) Function Tumor derived 5 × 10⁹ to 5 × 10¹⁰ CD45⁺, Active Tcells CD3⁺, Annexin-V-, DRAQ7- cells 20% Human 8.5% HSA W/V AdsorbtionSerum Albumin inhibitor Phosphate buffered 125 to 270 ml Isotonicdiluent Saline DMSO 10% V/V Cryoprotectant

With reference to FIG. 1 there is disclosed a disaggregation module ofthe device. The device may comprise a flexible container 1 a fordisaggregation and digestion in an embodiment involving enzymaticdigestion. An open end 1 b permits the transfer of solid tumor tissuematerial into the container 1 a. Hanging holes 1 c allow the container 1a to be hung and supported during transport or use. To maintain theaseptic conditions of the device, a target heat weld location 1 d allowsthe container 1 a to be sealed using a heat welder 13 c or othercomparable means. The container 1 a can have rounded edges 1 e oninternal surfaces of container 1 a to reduce losses, which may occur aspart of the transfer to examples illustrated in FIGS. 2 a-c or FIG. 3 aor FIG. 3 b . Tubing if enables media 3 a to be transferred intocontainer 1 a via sterile filter 2 a. Sterile filter 2 a comprises aspike to permit puncture of the seal in a subsequent module tofacilitate transfer of the media 3 a. Tubing 1 g enables digestionenzymes 3 b to be transferred into container 1 a via sterile filter 2 b.Sterile filter 2 b comprises a spike to permit puncture of a seal tofacilitate transfer of the digestive enzymes 3 b into the container 1 a.After disaggregation of the solid tumor tissue, especially involvingenzymatic digestion, the disaggregated mixture is transferred out oftubing 1 h via filter unit 4 a comprising sterile filter 4 b prior toentering a phase of incubation. Filter unit 4 a can be flexible topermit contortion without affecting the utility of the filtrationprocess. A filter 4 b removes the non-disaggregated tissue. Tubing clamp5 a allows the media 3 a to enter the flexible container 1 a via sterilefilter 2 a. In an embodiment involving enzymatic digestion, tubing clamp5 b allows the enzymes 3 b to enter the flexible container 1 a viasterile filter 2 b. Tubing clamp 5 c allows contents of flexiblecontainer 1 a to pass via filter unit 4 a into one or more examplesidentified in FIGS. 2 a-c or FIG. 3 a or FIG. 3 b.

According to FIG. 2 a , sterile filter 2 c permits the introduction ofmedia 3 a and/or a freezing solution 3 c required for cryopreservationof the disaggregated tumor tissue. Filter 4 d may be required foradditional size segregation of cell/tissue clumps. Filter 4 d isenclosed within filter unit 4 c, which can be flexible to permitcontortion without affecting the utility of the filtration process. Inan embodiment, a filter 4 e may be required to retain cells, but allowthe media and cell fragments to be washed out. Filter 4 d is similarlyenclosed within filter unit 4 c. In an embodiment, tubing clamp 5 d isin place to stop material from container 1 a that has passed throughfilter units 4 a and 4 c from returning back to container 1 a. In anembodiment, tubing clamp 5 e is in place to allow waste material fromcontainer 1 a that has passed through filter units 4 a, 4 c, and 4 e toenter waste container 6 a, but stops media 3 a or 3 c from entering viasterile filter 2 c. Tubing clamps 5 f stop material from container 1 athat has passed through filter units 4 a, 4 c, and 4 e from returning tothe source of the media 3 a or 3 c or transferring to one of theexamples illustrated in FIG. 3 a or FIG. 3 b before the waste has passedinto waste container 6 a via tubing clamp 5 e. Once the waste has beendepleted, tubing clamps 5 e and 5 d are closed and tubing clamps 5 fallow media 3 a or 3 c to transfer cells within filter 4 e into one ofthe examples illustrated in FIG. 3 a or FIG. 3 b . The waste container 6a has hanging holes to support the waste container 6 a during use and/ortransport.

FIG. 2 b illustrates the enrichment module of the device. Tubing clamp 5g allows the contents of container 1 a to enter flexible container 7 aof the enrichment module via filter unit 4 a. Tubing clamp 5 h allowscontents of container 7 a to pass through filter unit 8 a, retaining andenriching cells, while allowing waste and debris to pass through filter8 b into waste container 6 a with the pressure controlled by valve 8 cbefore the enriched cells return to container 7 a via open tubing clamp5 i. Tubing clamp 5 i allows contents of container 7 a via open tubingclamp 5 h to pass through filter unit 8 a, retaining and enriching cellswhile allowing waste and debris to pass through filter 8 b with thepressure controlled by valve 8 c before the enriched cells return tocontainer 7 a. After cell enrichment has occurred, tubing clamp 5 h isclosed and tubing clamp 5 j is opened to allow the contents of container7 a to pass to one of the examples illustrated in FIG. 3 a or FIG. 3 b .The waste container 6 a has hanging holes 6 b to support the wastecontainer 6 a during use and/or transport. Container 7 a of theenrichment module has hanging holes 7 b to support the container 7 aduring use and/or transport. The container 7 a can have rounded edges 7c on internal surfaces of container 7 a to reduce losses, which mayoccur as part of the transfer to examples illustrated in FIG. 3 a orFIG. 3 b . Tubing 7 d allows container 7 a to receive the contents ofcontainer 1 a via filter unit 4 a and filter unit 8 a. Tubing 7 e allowsthe contents of container 7 a to pass through filter unit 8 a, retainingand enriching cells while allowing waste and debris to pass throughfilter 8 b into waste container 6 a with the pressure controlled byvalve 8 c before the enriched cells return to container 7 a via opentubing clamp 5 i. Tubing 7 f allows the contents of container 7 a topass through filter unit 8 a, retaining and enriching cells whileallowing waste and debris to pass through filter 8 b into wastecontainer 6 a with the pressure controlled by valve 8 c before theenriched cells return to container 7 a.

FIG. 2 c illustrates another embodiment of the enrichment module. Tubingclamp 5 g allows the contents of container 1 a to enter the flexiblecontainer 7 a via filter unit 4 a. Tubing clamp 5 h allows contents ofcontainer 7 a to pass through filter unit 9 a, retaining and enrichingcells, while allowing waste and debris to pass through filter 9 b intowaste container 6 a with the pressure controlled by valve 9 c before theenriched cells return to container 7 a via open tubing clamp 5 i. Tubingclamp 5 i allows contents of container 7 a via open tubing clamp 5 h topass through filter unit 9 a, retaining and enriching cells whileallowing waste and debris to pass through filter 9 b with the pressurecontrolled by valve 9 c before the enriched cells return to container 7a. After cell enrichment has occurred, tubing clamp 5 h is closed andtubing clamp 5 j is opened to allow the contents of container 7 a topass to one of the examples illustrated in FIG. 3 a or FIG. 3 b . Thewaste container 6 a has hanging holes 6 b to support the waste container6 a during use and/or transport. Container 7 a of the enrichment modulehas hanging holes 7 b to support the container 7 a during use and/ortransport. The container 7 a can have rounded edges 7 c on internalsurfaces of container 7 a to reduce losses, which may occur as part ofthe transfer to examples illustrated in FIG. 3 a or FIG. 3 b . Tubing 7d allows container 7 a to receive the contents of container 1 a viafilter unit 4 a and filter unit 9 a. Tubing 7 e allows the contents ofcontainer 7 a to pass through filter unit 9 a, retaining and enrichingcells while allowing waste and debris to pass through filter 9 b intowaste container 6 a with the pressure controlled by valve 9 c before theenriched cells return to container 7 a via open tubing clamp 5 i. Tubing7 f allows the contents of container 7 a to pass through filter unit 9a, retaining and enriching cells while allowing waste and debris to passthrough filter 9 b into waste container 6 a with the pressure controlledby valve 9 c before the enriched cells return to container 7 a. Filterunit 9 a facilitates the filtration of the contents of container 7 a toremove waste media and debris via filter 9 b into waste container 6 awith the pressure controlled by valve 9 c before the enrich cells returnto container 7 a. Filter 9 b can be wound into a coil to increase thedistance that the waste must elute prior to reaching the waste container6 a for improved purification of the cell media, but facilitatetransport and storage of the improved filter 9 b.

FIG. 3 a illustrates an example of the stabilization module. Tubingclamp 5 k allows: the contents of container 1 a as illustrated in FIG. 1via filter unit 4 a, or as illustrated in FIG. 2 a via filter unit 4 c;or the contents of container 7 a as illustrated in FIG. 2 b via filterunit 8 a, or as illustrated in FIG. 2 c via filter unit 9 a to betransferred into container 10 a of the stabilization module. Container10 a of the stabilization module has hanging holes 10 b to support thecontainer 10 a during use and/or transport. The container 10 a can haverounded edges 10 c on internal surfaces of container 7 a to reducelosses, which may occur as part of the transfer out of tubing 10 e or 10f. Tubing 10 e enables the contents of container 10 a to be withdrawnvia connector 10 h. Tubing 10 f contains a flexible membrane to enable asterile spike to be introduced via an aseptic cover 10 g to enable thecontents of container 10 a to be withdrawn.

FIG. 3 b illustrates another embodiment of the stabilization module.Tubing clamp 5 l allows: the contents of container 1 a as illustrated inFIG. 1 via filter unit 4 a, or as illustrated in FIG. 2 a via filterunit 4 c; or the contents of container 7 a as illustrated in FIG. 2 bvia filter unit 8 a, or as illustrated in FIG. 2 c via filter unit 9 ato be transferred into container 11 a of the stabilization module.Container 11 a of the stabilization module has hanging holes 11 b tosupport the container 10 a during use and/or transport. The container 10a can have rounded edges 10 c on internal surfaces of container 7 a toreduce losses, which may occur as part of the transfer out of tubing 11f. Tubing clamp 5 m allows media 3 c to enter the flexible container 11a via sterile filter 2 c. Tubing clamp allows the contents of container11 a to enter one of the cryopreservation containers 12 a depending onthe open or closed status of tubing clamps 5 o, 5 p, 5 q, 5 r, 5 s, and5 t. Tubing clamps 5 p, 5 q, 5 r, 5 s, and 5 t allow the contents ofcontainer 11 a to enter one of the cryopreservation containers 12 a.Tubing 11 d enables container 11 a to receive: the contents of container1 a as illustrated in FIG. 1 via filter unit 4 a, or as illustrated inFIG. 2 a via filter unit 4 c; or the contents of container 7 a asillustrated in FIG. 2 b via filter unit 8 a, or as illustrated in FIG. 2c via filter unit 9 a. Tubing 11 e allows cryopreservation media 3 c tobe transferred into container 11 a. Tubing 11 f enables the contents ofcontainer 11 a to be transferred to cryopreservation containers 12 a,where the final disaggregated UTIL product as a single cell suspensionis stored for future use in the rapid expansion process.Cryopreservation containers 12 a have a fixtures 12 b to allow aseptictransfer of the TILs out of the cryopreservation containers 12 a.Cryopreservation containers 12 a have a space 12 c that is suitable forthe volume of the UTIL cell suspension to be stored. Thecryopreservation containers 12 a also have a target location 12 d forwelding the tubing 11 f to the cryopreservation containers 12 a.

FIG. 4 illustrates another example of the device and kit. Pegs 13 aallow the media 3 a, 3 b, and 3 c to be hung. Pegs 13 b are connected toweight sensors for hanging container 1 a and depending on the embodimentutilized, could include one or more of containers 7 a, 10 a, and/or 11a. The weight sensors are used to define decision stages to control theautomated processing of the materials. A heat welder 13 c can be used toseal container 1 a at the target site following the introduction of theresected solid tumor tissue into container 1 a. The disaggregationmodule 13 d has an opening that can be closed and locked to enabledisaggregation and can control the temperature to be between 0° C. and40° C. to a tolerance of 1° C. to enable digestion where digestiveenzymes are used for disaggregation of the solid tumor tissue. Thedisaggregation module 13 d also has a built in sensor to assess thelevel of solid tissue disaggregation by determining the variation inlight distribution against time to identify change and thereby identifycompletion of the disaggregation process, which occurs over a period ofseconds to hours. Disaggregation module 13 d may also comprisedisaggregation surfaces 13 f, which come directly into contact withcontainer 1 a and pushes against the back of the disaggregation module13 d enclosure, which can be closed and locked during disaggregation anddigestion where enzymes are utilized. A final formulation module 13 ehas an enclosure that allows temperature control of either containers 10a or 11 a depending on the embodiment utilized, which is capable ofcontrolling temperatures between 0° C. and ambient environmentaltemperature to a tolerance of 1° C. Tubing clamps 13 g and 13 j act asinput and output ports, disposed within tubing locators 131, andfacilitate transport of the disaggregated tumor product between thecontainers 1 a, 10 a, or 11 a depending on the embodiment utilized.Peristaltic tubing pumps 13 h control the transfer of the media 3 a or 3c between the tubing clamps 13 g and 13 j that act as input and outputports. Tubing valve 13 k assists in controlling the pressure via valves8 c and 9 c in the enrichment module as illustrated in FIGS. 2 b and 2 c. Pegs 131 allow for the hanging of waste container 6 a and/orcryopreservation containers 12 a depending on the embodiment utilized.The embodiment can also include a tubing welder 13 m required forconnecting the cryopreservation containers 12 a to the device asillustrated in FIG. 3 b . The embodiment can also include a tubingcutter 13 n for disconnecting the cryopreservation containers 12 a tothe device as illustrated in FIG. 3 b . Controlled rate cooling module13 o is capable of cooling or maintaining any temperature between 8° C.and at least −80° C. to assist in the cryopreservation process.

The method of the invention is exemplified according to the followingprocess. It is clearly stated that other than the essential features ofthe method, the various optional steps listed herein can beindependently combined to achieve the relevant technical advantagesassociated with the type of sampling and result to be achieved.

A semi-automatic aseptic tissue processing method comprises:automatically determining aseptic disaggregation tissue processing stepsand one or more further tissue processing steps and their associatedconditions from a digital tag identifier on an aseptic processing kit,optionally in accordance with the kit described herein; placing a tissuesample into a flexible plastic container of the aseptic processing kit;and processing the tissue sample by automatically executing the one ormore tissue processing steps by communicating with and controlling thedisaggregation module; the optional enrichment module; and thestabilization module.

Essentially the process may comprise taking an open ended bag (firstflexible container that is part of disaggregation module) that willreceive the biopsy/tissue sample, preferably a resected tumor, which isalready connected via one or more conduits to or can be connected via amanual operator controlled aseptic connection to

I. a single container with digestion media (second flexible containerthat is part of the disaggregation module) and with or without astabilization solution (same second flexible container is part of thestabilization module also)

II. one container with a digestion solution (second flexible containerthat is part of the disaggregation module) and another container with astabilization solution (fourth flexible container is part of thestabilization module)

on addition of the biopsy and sealing of the open ended bag thedigestion media can be added via the conduit or aseptic connections(conduit/ports claim 1) and the tissue material processed.

On completion of the digestion by which point the tissue is now a singleor small number aggregate cellular suspension the cells can optionallybe filtered prior to step 4 (optional enrichment module for filtrationcomprises the first flexible container containing sample and filtered toa third container for receiving the enriched filtrate).

Where the stabilization media is not present in the same flexiblecontainer, the container with stabilization solution is added by openingthe attached conduit or manual operator controlled asepticallyconnection to be competed and said connection to be opened enabling inboth cases the stabilization solution to be added before the processcontinues.

The single or small number aggregate cellular suspension in the originalflexible container or which may be optionally subdivided into multiplestorage stabilization containers thereafter are maintained in a stablestate on the device and/or will undergo cryopreservation prior toremoval for, transport, storage and or used in their ultimately utility.The stabilization module also comprises first or third container as usedin storage/freezing/storage.

In one further non-limiting example of the process:

a) Collection of tissue sample by a separate procedure such as a biopsyor surgery to collect the required tissue material (not part of theinvention) is placed into the initial flexible plastic container (seee.g., FIG. 1 , container 1 a).

b) Media (see e.g. FIG. 1 , media 3 a) is transferred into thedisaggregation chamber, or in one example also enters and collectsenzymes (see e.g. FIG. 1 , enzymes 3 b), prior to disaggregation usingone or more of the following examples of the invention a mechanism suchas weight sensors (see e.g. FIG. 1, 13 b as part of module 13 d)assesses the required amount of media to add either determined by:direct operator input or weight of solid tissue.

c) The single use flexible disaggregation container, solid tissue, mediaand in one example enzymes are combined during a physical disaggregationprocess for a minimum of a few seconds up to several hours with anoptimal time of between 1 and 10 minutes required to break up the solidtissue until there is no visual change (See FIG. 5 and Table 1). Thedisaggregation device is designed to compress the tissues using avariable speed and time depending upon the time taken to disaggregateand feedback via sensors within the disaggregation module (see FIG. 1,13 d).

d) In one embodiment where enzymes are present this will requireincubation periods at an optimal temperature of between 30 and 37° C.but could be as low as 0° C. up to 40° C. for at least 1 minute toseveral hours but more preferable 15 to 45 minutes.

e) Step c and in the embodiment where enzymes step d) can be repeateduntil the tissue stops changing or the see example has beendisaggregated into a liquid cell suspension whichever comes 1stmonitored by a sensor in the disaggregation module disaggregation module(see FIG. 1, 13 d).

1) In one embodiment incompletely disaggregated tissues, associatedmaterial and impurities are removed enabling enrichment of the cellsuspension by passing the disaggregated tissue and media using one ormore of the following embodiments:

i. Direct pass through one or more mechanical filters with holes atleast >0.1 μm to 1000 μm but most preferably between 50 and 250 μm andmore preferably 100 μm to 200 μm (illustrated in FIG. 2 a ).

ii. Density based separation using centrifugation and/or sedimentationwith or without a cell aligned density retention solution (e.g.Ficoll-paque GE Healthcare).

iii. Hydrodynamic filtration where fluid flow and flow obstructingmaterials enhance the resolution and fractionation of the cells andimpurities based on size and shape

iv. Field flow fractionation where an applied field (e.g. flow,electric, gravitational, centrifugal) acts in a perpendicular or reversedirection to the selection flow (e.g. Tangential flow filtration, Hollowfiber flow filtration, Asymmetric flow filtration, Centrifugal flowfiltration). In which case: cells or impurities which are mostresponsive to the force are driven to the wall where flow is lowest andtherefore a long retention time; while cells or impurities which areleast responsive to the force remain laminar to the flow and elutequickly (tangential flow filtration illustrated in FIGS. 2 b and c ).

v. Acoustophoresis where one or more an acoustic frequency(ies) tuned toor harmonized with populations of cells or impurities is used to drivethe required cells or impurities in a tangential path to the inputstream.

g) In one embodiment the disaggregated enriched tissue product will beresuspended in a fresh media (FIG. 2 a using media 3 a) such as:

i. a cell enrichment media in order to undergo an independent targetedenrichment procedure as described previously

ii. direct cell culture or cold storage media (such as HypoThermosol®from BioLife Solutions.

h) in the embodiment employed in g) the resuspended disaggregated solidtissue derived product is transferred to one of the embodiment finalproduct containers (illustrated in FIG. 3 a ) for storage for hours todays prior to being used for its ultimate utility.

i) otherwise after step f) the embodiment applies (illustrated in FIG. 3h ) will apply where the disaggregated solid tissue derived productundergoes re-suspension in a cryoprotectant (FIG. 3 b , media 3 c) afreezing solution for storage of the disaggregated solid tissue derivedproduct for days to years such as CryoStor® Freezing solution fromBioLife Solution.

j) At this stage the disaggregated solid tissue derived product isre-suspended in freezing solution (FIG. 4 , module 13 e) and transferredto one or more flexible cryopreservation container(s) (illustrated inFIG. 3 a , container 12 a) and in one embodiment of the device there isa controlled rate freezing process (FIG. 4 , module 13 o).

k) After which the bags can be separated from the device and asepticprocessing kit for independent storage or distribution.

In further embodiments, a disposable kit of the invention can be usedwith an automatic device for semi-automatic aseptic processing of tissuesamples. FIGS. 6 and 7 depict disposable kits of the invention.

FIG. 6 depicts a semi-automatic aseptic tissue processing method usingmultiple flexible containers for different starting solutions that arepart of the modules of the process used for disaggregation andstabilization.

Process step 1—The user may login to device and scan the tag on theaseptic kit using the device to transfer the automatic processing stepsto be used. The device processor recognizes the tag and is provided withinformation needed to carry out the specific processing instructionsrelated to that particular kit.

Process step 2—The digestion media containing flexible hag (part ofdisaggregation module) and cryo/stabilization solution containingflexible bag (part of the stabilization module) are each hung or securedto the device.

Process step 3—The biopsy or tissue sample for processing may be placedinto a flexible container (part of both modules) of the aseptic kit viaan open end.

Process step 4—The flexible container comprising the sample may then besealed using a heat weld to close the open end (used to add the sampleduring initial processing).

Process step 5—The user may then interact with the user interface of theprocessor to confirm the tissue sample is present and enter any furthertissue material specific information, if required.

Process step 6—Digestion media and cryo/stabilization solution flexiblecontainers are connected with the flexible container housing the sample,after which it may be placed into the device for automatic processing.

Process step 7—The device executes the cycles according to the kitinformation undertaking disaggregation of the sample andstabilization/cryo preservation of resulting cells.

Process step 8—When stabilized/frozen disconnect and discard used mediaand cryo/stabilization containers of kit. Tissue processed into singleor multi-cell solution in flexible container is disconnected beforetransferring into storage or transport container prior to its ultimateutilization.

In another embodiment, FIG. 7 depicts flexible containers comprising themedia used in the process may be shared between the modules of theaseptic processing kit and method.

Process step 1—The user may login to device and scan the tag on theaseptic kit using the device to transfer the automatic processing stepsto be used.

Process step 2—A flexible bag (part of disaggregation/stabilizationmodule) comprising both the media and cryo/stabilization solution ishung or otherwise secured to the device.

Process step 3—The biopsy or tissue sample for processing may be placedinto a further flexible container (part of both modules) of the aseptickit via an open end.

Process step 4—The flexible container comprising the sample may then besealed using a heat weld to close the open end.

Process step 5—The user may then interact with the user interface of theprocessor to confirm the tissue sample is present and enter any tissuematerial specific information, if required.

Process step 6—Digestion media and cryo/stabilization solution flexiblecontainer is connected with the flexible container housing the sample,after which it may be placed into the device for automatic processing.

Process step 7—The device cycles to enable disaggregation of the sampleand stabilization of resulting cells, optionally via cryopreservation.

Process step 8—When freezing/stabilizing is complete the userdisconnects and discard used flexible containers of kit. Tissueprocessed into single or multi-cell solution in the remaining flexiblecontainer is disconnected before transferring into storage or transportcontainer prior to its ultimate utilization.

By way of example, in another embodiment of the method of the invention,where the disaggregation process is being supplemented with enzymaticdigestion the media formulation for enzymatic digestion must besupplemented with enzymes that aid in protein breakdown causing the cellto cell boundaries to breakdown as described above.

Various liquid formulations known in the art of cell culturing or cellhandling can be used as the liquid formulation used for celldisaggregation and enzymatic digestion of solid tissues, including butnot limited to one or more of the following media Organ PreservationSolutions, selective lysis solutions, PBS, DM EM, HBSS, DPBS, PM I,Iscove's medium, XVIVO™, AIM-V™, Lactated Ringer's solution, Ringer'sacetate, saline, PLASMALYTE™ solution, crystalloid solutions and IVfluids, colloid solutions and IV fluids, five percent dextrose in water(D5W), Hartmann's Solution DM EM, HBSS, DPBS, RPMI, AIM-V™, Iscove'smedium, XVIVO™, each can be optionally supplemented with additional cellsupporting factors e.g. with fetal calf serum, human serum or serumsubstitutes or other nutrients or Cytokines to aid in cell recovery andsurvival or specific cell depletion. The media can be standard cellmedia like the above mentioned media or special media for e.g. primaryhuman cell culture (e.g. for endothelia cells, hepatocytes orkeratinocytes) or stem cells (e.g. dendritic cell maturation,hematopoietic expansion, keratonocytes, mesenchymal stem cells or Tcells). The media may have supplements or reagents well known in theart, e.g. albumins and transport proteins, amino acids and vitamins,metal-ion(s), antibiotics, attachments factors, de-attachment factors,surfactants, growth factors and cytokines, hormones or solubilizingagents. Various media are commercially available e. g. fromThermoFisher, Lonza or Sigma-Aldrich or similar media manufacturers andsuppliers.

The liquid formulation required for enzymatic digestion must havesufficient calcium ions present in the of at least 0.1 mM up to 50 mMwith an optimal range of 2 to 7 mM ideally 5 mM.

The solid tissue to be digested can be washed after disaggregation witha liquid formulation containing chelating agents EGTA and EDTA to removeadhesion factors and inhibitory proteins prior to washing and removal ofEDTA and EGTA prior to enzymatic digestion.

The liquid formulation required for enzymatic digestion is more optimalwith minimal chelating agents EGTA and EDTA which can severely inhibitenzyme activity by removing calcium ions required for enzyme stabilityand activity. In addition, b-mercaptoethanol, cysteine and8-hydroxyquinoline-5-sulfonate are other known inhibitory substances.

As described in preferred embodiments the final cell container forcryopreservation is a flexible container manufactured from resilientdeform able material. In this embodiment of the device the finalcontainer is either transferred directly to a freezer −20 to −190° C. ormore, optimally located in the controlled rate freezing apparatus eitherassociated with the device or supplied separately (manufactured by forexample Planer Products or Asymptote Ltd) in which the temperature ofthe freezing chamber and the flexible storage container(s) employed tocontain the enriched disaggregated solid tissue container is controlledeither by: injecting a cold gas (normally nitrogen for example Planerproducts); or by removing heat away from the controlled coolingsurface(s). Both methods result in the ability to accurately controlwith an error of less than 1° C. or more preferable 0.1° C. the freezingprocess at the required rate for the specific cell(s) to be frozen basedon the freezing solution and the desired viability of the product. Thiscryopreservation process must take into account the ice nucleationtemperature which is ideally as close as possible to the meltingtemperature of the freezing solution. Followed by crystal growth in anaqueous solution, water is removed from the system as ice, and theconcentration of the residual unfrozen solution increases. As thetemperature is lowered, more ice forms, decreasing the residualnon-frozen fraction which further increases in concentration. In aqueoussolutions, there exists a large temperature range in which ice co-existswith a concentrated aqueous solution. Eventually through temperaturereduction the solution reaches the glass transition state at which pointthe freezing solution and cells move from a viscous solution to a solidlike state below this temperature the cells can undergo no furtherbiological changes and hence are stabilized, for years potentiallydecades, until required.

The disaggregated cell products achieved by the method of the presentinvention can be cultured and/or analyzed (characterized) according toall methods known to the person skilled in the art.

The TILs obtainable by the methods disclosed herein may be used forsubsequent steps such as research, diagnostics, tissue-banks, biobanks,pharmacological or clinical applications known to the person skilled inthe art. TILs can then be taken into culture using a Medium optimizedfor this application, e.g. T cell Mixed Media (Cellular Therapeutics)usually containing but not limited to growth factors such as IL-2, IL-7,IL-15, IL-21 or stimulatory conditions such as plates or polystyrenebeads coated with antibodies. In the present invention isolated cellswere seeded into culture containers and maintained using proceduresstandardly used by a person skilled in the art such as a humidifiedatmosphere (1-20% usually 5% CO, 80 to 99% usually 95% air) attemperatures between 1 to 40° C., usually 37° C., for several weeks andsupplements may be added supplemented with 10% FBS and 3000 IU/mL IL-2.

The enriched TILs could be used before and/or after cell culturing as apharmaceutical composition in the therapy, e.g. cellular therapy, orprevention of diseases. The pharmaceutical composition can be used forthe treatment and/or prevention of diseases in mammals, especiallyhumans, possibly including administration of a pharmaceuticallyeffective amount of the pharmaceutical composition to the mammal.

Such TIL cultures, in addition to being formulated as a drug product forthe treatment of various cancers, can be used to study e.g. cellfunction, tumor cell killing, cell signaling, biomarkers, cell pathways,nucleic acids, and other cell or tissue related factors that may be usedto identify donor, tissue, cell or nucleic acid status.

The disease may be any disease, which can be treated and/or preventedthrough the presence of solid tissue derived cells and/or throughincreasing the concentration of the relevant cells in/at the relevantplace, i.e. the tumors or sites of disease. The treated and/orpreventively treated disease may be any disorder, e.g. cancer or adegenerative disorder. The treatment may be the transplantation ofenriched, engineered or expanded cells or any combination of these andeither administered to the relevant part of the body or suppliedsystemically.

Pharmaceutical compositions of the present disclosure may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials.

As described herein the invention provides a kit that allows for thereceipt, processing, storing, and/or isolating of material such astissue, in particular mammalian tissue. Further, the invention providescomponents of the kit such as flexible containers, for example bags,filters, valves, brackets, clamps, connectors, and/or conduits such astubing. In particular, bags may be coupled to one or more tubes orsections of tubing adapted to enable flow of tissue material betweenvarious components of a cryopreservation kit.

Processing of tissue to cells using a cryopreservation kit and/or acollection bag may include automated and/or semi-automated devices andmethods.

Moreover, by utilizing the bags, kit, devices and processes describedherein, in conjunction with ordinary skill in the art, furtherembodiments of the present disclosure can be readily identified. Thoseskilled in the art will readily understand known variations.

Design Patent Application Ser. No. 29/740,293 provides a tissuecollection bag suitable for tissue collection. The top of the tissuecollection bag of the invention is open, for receiving tissue, e.g., atissue biopsy, such as animal (e.g., domestic animal such as dog or cat)or human cancerous tissue. The tissue collection bag is to be sealedwith collected tissue therein, and for the tissue so sealed therein tobe processed therein, e.g., processing can include agitation and/orcompression, e.g., gentle agitation and/or compression, and/or enzymaticdigestion of the tissue therein. Advantageously the tissue processingand extraction therein, from the desired material, such as tumorinfiltrating lymphocytes (TILs), can be in a closed system. Advantageousor preferred embodiments can include indicia to indicate the patientfrom whom the tissue was collected and/or indicia to show where thecollection bag may be clamped or affixed in place in an instrument forapplying agitation and/or indicia to show where the collection bag maybe sealed, e.g., by heat sealing (which may be part of the instrumentfor processing). Advantageously, prior to application of processing, thecollection bag is clamped or affixed into an instrument for processingand/or sealed, e.g., heat sealed. In certain illustrations, tubing maybe shown with dotted lines or stippling to show that the tubing is notnecessarily considered part of the inventive design; but in certainembodiments may be considered part of the inventive design. The dottedlines or stippling is to be interpreted as the tubing may be present orabsent and may be claimed as either or both, i.e., throughout thedrawings the tubing can form part of the inventive design (and also maynot necessarily be part of the inventive design). In addition, whilecertain illustrations show no indicia, indicia that may indicate apatient from whom a sample was obtained, indicia that may indicate apatient from whom a sample was obtained and where the tissue collectionbag may be clamped or affixed into an instrument, and indicia that mayindicate a patient from whom a sample was obtained and where the tissuecollection bag may be clamped or affixed into in an instrument and wherethe tissue collection bag may be sealed, e.g., heat sealed, it is to beunderstood that the inventive design can include variations thereof,e.g., the inventive design may include indicia that may indicate apatient from whom a sample was obtained and where the tissue collectionbag may be heat sealed without also indicia showing where the tissuecollection bag may be clamped or affixed into an instrument; and theinventive design may include indicia that may indicate where the tissuecollection bag may be heat sealed and/or indicia showing where thetissue collection hag may be clamped or affixed into an instrument butwithout indicia indicating a patient from whom a sample was obtained(including as patient indicia may be imprinted onto the tissuecollection bag as it is being used, whereas indicia as to clamping oraffixing or heat sealing may already be on the tissue collection bagprior to being in use). The tissue collection bag including anyassociated tubing can be generally clear or transparent or translucent,or any color desired. The tissue collection bag including any associatedtubing can be generally fabricated in ways analogous to the fabricationof: closed or sealed, blood collection, tissue culture, bio-processingor cryopreservation bags and associated tubing. The associated tubing inthe invention may be constructed from any desired material, withpolyvinyl chloride (PVC) or a material including PVC as a desiredmaterial as that is advantageous for welding and/or sealing. The portionof the tissue collection bag of the invention for receiving the tissuecan be made from any desired material, with ethylene vinyl acetate (EVA)or a material including EVA as a desired material as that isadvantageous for heat sealing.

As shown in FIG. 11A, an embodiment for kit 2 for treating tissue, forexample, the disaggregation, enrichment, and/or stabilization of tissue.Tissue to be treated may include solid eukaryotic, in particular,mammalian tissue, such as tissue from a sample and/or a biopsy. Kit 2includes components such as bags 4, 6, such as collection bag 4 andcryopreservation bag 6. Kits as depicted in FIG. 11A-D may be used in anautomatic or a semi-automatic device for treatment.

In some embodiments, kit components may include indicators, such ascodes, letters, words, names, alphanumeric codes, numbers, images, barcodes, quick response (QR) codes, trackers such as smart trackers and/orBluetooth trackers, tags such as a radio frequency tag, and/or otherdigitally recognizable identification tag so that it may be scanned andrecognized during automated and/or semi-automated treatment such aswithin an automated device in embodiments of the present invention. Forexample, a tag may provide information about the conditions and/or stepsrequired to be automatically treated. For example, scanning a kitcomponent such as a bag may allow an automated system used with the kitto treat tissue without further intervention and/or contamination. Inparticular, a tissue sample that has been placed in a collection bag fortreatment in a disaggregation element of a device. The collection bagmay be sealed before treatment begins. In some embodiments, a collectionbag may be sealed manually and/or automatically using energy such asheat, radio frequency energy, high frequency (HF) energy, dielectricenergy, and/or any other method known in the art before treatmentbegins.

In some embodiments, a heat sealer (e.g., Van der Staehl MS-350, UlineH-190 Impulse Sealer, or similar sealers known in the art) with aheating bar the bar may be used to create a seal on a bag.

In a particular embodiment, when using a heat sealer it may beadvantageous to form the seal at a temperature below about 100° C. andin at a pressure in a range from about 0.8 bar to about 2.8 bar. Thiselevated temperature and pressure may be applied for about eight secondsafter which the temperature may be reduced but the pressure continues tobe applied for about 2 to 3 seconds in some embodiments. The values fortemperature, pressure, and time will vary based upon the formulation ofthe material forming the bag and in particular the material forming theseal. For example, another material may require that the sealer reach atemperature above about 210° F. (98.9° C.) for a minimum of about 3seconds after which the heating bar may be allowed to cool for 5 secondsprior to removing the heating bar.

Positioning of the material to be sealed may be critical to the strengthof the seal formed. For example, incomplete seals, folds, channels,and/or gaps in the material to be sealed may reduce the strength of theseal.

Seals may be tested for strength using a seal peel test (i.e., ASTMF88/F88M), and/or a burst test (i.e., ASTM F1140/F1140M or ASTMF2051/F2054M).

In some embodiments, a bag or a flexible container may withstand a forceof 100 Newtons during use when properly sealed and further secured witha clamp when positioned within a device for treatment and/or processing.A bag or a flexible container embodiment may be constructed to withstanda force of 75 Newtons during use when properly sealed and furthersecured with a clamp when positioned within a device for treatmentand/or processing.

As shown in FIG. 11A, kit 2 includes disaggregation element 4 wherecollection bag 5 may be treated, enrichment element 8 where filter 9 maybe located, and stabilization element 6 where cryopreservation bag 7 isused to preserve the desired material. In a component of kit 2, such ascollection bag 5, tissue is treated. For example, collection bag 5 maybe used for the disaggregation of solid tissue derived from eukaryoticcells. Tissue may be treated in such manner such that a majority of theresulting tissue after processing may be single cells and/or small cellnumber aggregates. Further, processing may occur in the kit and/or inthe collection bag in particular.

Enrichment of the treated tissue may occur at enrichment element 8 infilter 9. Filter 9 may be selected such that the filtered composition(i.e., desired material) entering tubing 11 may have constituents havinga predetermined size. Filter 9 may be selected such that the desiredmaterial composition entering tubing 11 may have constituents such astumor infiltrating lymphocytes (TILs) having an average size of lessthan about 200 pm. In particular, in an embodiment the desired materialmay include tumor infiltrating lymphocytes (TILs) having an average sizeof less than about 170 pm.

In some embodiments, the desired material may include tumor infiltratinglymphocytes (TILs) in a range from about 15 pm to about 500 pm. Forexample, filter 9 may, in an embodiment, be configured such that atissue composition entering tubing 11 has constituents having an averagesize of less about 200 pm. In particular, the desired material exitingthe filter and entering the tubing 11 after being filtered may haveconstituents having an average size of less than about 170 pm.

In some embodiments, filter 9 is configured such that the filteredcomposition entering tubing 11 has constituents having a size in a rangefrom about 50 pm to about 300 pm. For example, filter 9 may in anembodiment be configured such that a tissue composition entering tubing11 has constituents having an average size in a range from about 150 pmto about 200 pm.

As shown in FIG. 11A, stabilization element 6 of the system for treatingtissue is where cryopreservation bag 7 may be used to stabilize thetissue composition for storage and/or transport.

FIG. 11B depicts kit 2 having valves 12, 13. Valves may be needle freevalves. Valves 12, 13 may be used to provide enzyme media such as atumor digesting media, cryoprotectant, and/or cryopreservation media. Inparticular, valve 12 may be used to provide an enzyme media to tubing10. Enzyme media may travel to collection bag 4 to aid in the processingof tissue placed in bag 5.

Valve 13 may be used to provide a cryoprotectant such as a DMSO solutionto tubing 11 such that the DMSO solution may travel to cryopreservationbag 7. In some embodiments, a cryoprotectant such as a DMSO solution maymix with the filtered material entering tubing 11 such that a combinedcomposition of DMSO solution and filtered material enterscryopreservation bag 7. The filtered material entering tubing 11 mayinclude constituents, such as tumor infiltrating lymphocytes (TILs)having a predetermined average size. For example, in some embodiments anaverage size of constituents in the filtered composition may be lessthan about 200 pm.

In some embodiments, as shown in FIG. 11C, kit 2 includes clamps 14around filter 9 to ensure that materials provided through valves 12, 13are inhibited and/or prevented from flowing into filter 9. Valve 13 maybe used to provide a cryoprotectant to tubing 11 such that thecryoprotectant may mix with the filtered material entering tubing 11from filter 9. For example, clamp 14 may be positioned to inhibit and/orprevent flow of the cryoprotectant in the direction of filter 9. In someembodiments, after the filtered solution starts to flow from filter 9clamp 14 will be released such that a combined composition ofcryoprotectant and filtered material enters cryopreservation bag 7 atstabilization element 6. The filtered material entering tubing 11 mayinclude constituents, such as tumor infiltrating lymphocytes (TILs)having a predetermined average size. For example, in some embodiments anaverage size of constituents in the filtered composition may be lessthan about 200 pm.

An embodiment of kit 2 may include ports 16 on cryopreservation bag 7 asis shown FIG. 11D. Ports may be used to add and/or remove materials fromcryopreservation bag 7. For example, test samples may be removed fromcryopreservation bag.

FIG. 12A shows a perspective view of an embodiment of bag 22 for use ina kit. Bag 22 may include connector 24, open section 26, sealed section21, and positioners 23. Connector 24 may be used to couple bag 22 totubing 25. Positioners 23 may be openings in bag 22.

Bags, such as collection bags and/or cryopreservation bags, and anyassociated tubing may be generally clear, transparent, translucent, anycolor desired, or a combination thereof. Bags, for example, collectionbags and/or cryopreservation bags, and/or tubing may be generallyfabricated in ways analogous to the fabrication of closed and/or sealedblood and/or cryopreservation bags and the associated tubing.

Bags for use in the invention described herein include a collection bagand a cryopreservation bag may include at least a portion made from apredetermined material such as a thermoplastic, polyolefin polymer,ethylene vinyl acetate (EVA), blends such as copolymers, for example, avinyl acetate and polyolefin polymer blend (i.e., OriGen Biomedical EVOfilm), a material that includes EVA, and/or coextruded layers ofsealable plastics.

Materials for use in the bag may be selected for a specific propertyand/or a selection of properties, for example, sealability such assealability due to heat welding, or use of radio frequency energy, gaspermeability, flexibility for example low temperature flexibility (e.g.,at −150° C., or −195° C.), elasticity for example low temperatureelasticity, chemical resistance, optical clarity, biocompatibility suchas cytotoxicity, hemolytic activity, resistance to leaching, having lowparticulates, high transmissions rates for particular gases (e.g.,Oxygen and/or Carbon dioxide), and/or complying with regulatoryrequirements. For example, materials used in the bag may be selected forhaving a tensile strength greater than about 2500 psi (172 bar) whentested according to the test method for tensile strength outlined inASTM D-638. In particular, an embodiment of a flexible container, suchas a bag, have use materials having a tensile strength greater thanabout 2800 psi (193 bar) when tested according to the test method fortensile strength outlined in ASTM D-638.

In some embodiments, materials may be selected for specific propertiesfor use in a coextruded material to form at least one layer of a bag.Layers may be constructed such that when constructed an interior layerof the bag is relatively biocompatible, that is the material on an innersurface of the bag is stable and does not leach into the contents of thebag.

For example, a property of interest that may be used to select amaterial for kit component such as a collection bag, a cryopreservationbag, and/or the associated tubing may relate to sealing, for exampleheat sealing.

Seals may be tested for strength using a seal peel test (i.e., ASTMF88/F88M), and/or a burst test (i.e., ASTM F1140/F1140M or ASTMF2051/F2054M).

In some embodiments, a bag or a flexible container may withstand a forceof 100 Newton's during use when properly sealed and further secured witha clamp when positioned within a device for treatment and/or processing.A bag or a flexible container embodiment may be constructed to withstanda force of 75 Newtons during use when properly sealed and furthersecured with a clamp when positioned within a device for treatmentand/or processing.

Dimensions of bags, in particular collection bags and/or preservativebags, may be specific to the device used to conduct treatment and/orprocessing. Bag size should be adjusted based on the configurationand/or size of the device(s) used to conduct treatment. Particular careshould be taken with placement and/or size of any component that extendsbeyond the border of a bag, for example, a port, connector or the like.Components such as ports may interfere with the operation of a deviceused to conduct treatment and/or processing. Further, care should betaken to ensure that a thickness of bags comports with the requirementof the machine, in particular with respect to sealed material such asthe manufactured seal.

Tubing in the invention may be constructed from any desired materialincluding, but not limited to polyvinyl chloride (PVC). For example, PVCmay be a desired material as PVC is advantageous for welding and/orsealing.

In some embodiments, as depicted in FIGS. 12A-12E, 13A-13E, 14, 20A-20E,21A-21E, 22A-22D, 27A, 28, 33, and 34 at least one end of a collectionbag may be open for receiving tissue. In particular, in an embodiment, atissue sample, for example from a biopsy may be placed in the bagthrough the open end, for example, a top end. In some cases, the biopsysample may be cancerous tissue from an animal (e.g., domestic animalsuch as dog or cat) or a human.

As shown in FIG. 12A, bag 22 may be used as a tissue collection bag. Forexample, after tissue is positioned in the bag, the bag may be sealed,and then may be processed. Processing may include agitation, e.g.,gentle agitation, extraction, and/or enzymatic digestion of the tissuein the bag. Tissue processing and extraction therefrom of desiredmaterial, such as tumor infiltrating lymphocytes (T1Ls), can be in aclosed system. Advantageous or preferred embodiments may includeindicators to indicate the patient from whom the tissue was collectedand/or marks to show where the collection bag may be clamped, sealed,acted upon by a device, and/or affixed in place in an instrument.

In some embodiments, bag 22 may be formed from a sealable material. Forexample, bag 22 may be formed from materials including, but not limitedto polymers such as synthetic polymers including aliphatic orsemi-aromatic polyamides (e.g., Nylon), ethylene-vinyl acetate (EVA) andblends thereof, a vinyl acetate and polyolefin polymer blend,thermoplastic polyurethanes (TPU), polyethylene (PE) and/or combinationsof polymers. Portions of a bag may be sealed and/or welded with energysuch as heat, radio frequency energy, high frequency (HF) energy,dielectric energy, and/or any other method known in the art.

A collection bag may be used as a processing and/or disaggregation bag.Collection bags may have width in a range from about 4 cm to about 12 cmand a width in a range from about 10 cm to about 30 cm.

For example, a collection bag for use in processing may have a width ofabout 7.8 cm and a length of about 20 cm. In particular, a bag may beheat sealable, for example, using an EVA polymer and blends thereof, avinyl acetate and polyolefin polymer blend, and/or one or morepolyamides (Nylon).

As depicted in FIG. 12A, bag 22 may be used as a tissue collection bagfor sealing tissue therein for processing of the invention.

FIG. 12B shows a perspective view of an embodiment of hag 22 for use asa tissue collection bag. Tissue may be sealed in the bag and thenprocessed. Bag 22 as shown in FIG. 12B may be marked with indicators 27,28, such as a patient identifier that can identify a patient from whom atissue sample or biopsy has been taken or obtained.

Indicators may include, but are not limited to codes, letters, words,names, alphanumeric codes, numbers, images, bar codes, quick response(QR) codes, tags, trackers such as smart tracker tags or Bluetoothtrackers, and/or any indicator known in the art. In some embodiments,indicators may be printed on, etched on, and/or adhered to a surface ofa component of a kit. For example, indicators may be printed directly ona surface of at least one component of a kit as shown in FIG. 12B.Indicators may also be positioned on a bag using an adhesive, forexample, a sticker or tracker may be placed on a bag and/or on multiplebags. For example, as shown FIG. 12B bag 22 includes multiple indicators28 (numeric code), 27 (QR code).

FIG. 12C shows a perspective view of a bag for use as a tissuecollection bag. Tissue may be inserted into bag 22 for processing.Indicators may be used to can identify a patient from whom a tissuesample and/or biopsy has been taken or obtained. As shown in FIG. 12C,indicators 27, 28 include a QR code and identifying number used to tracka sample, locate a sample, and/or track status of a sample in a process.For example, in some embodiments indicators may be used locate a sampleat any given position in a laboratory. Indicators may be placed on bagprior to and/or during use, for example, as the bag is being taken outfor use with a sample, patient indicators may be imprinted onto the bag.Further, bag 22 may include mark 29. Marks may be used to show whereseals, clamps, and/or instruments should be positioned.

Indicators, for example QR codes, tags such as smart tags, and/ortrackers may be used to identify a sample within a bag as well as toinstruct a device's processor such that the device runs a specificprogram according to a type of disaggregation, enrichment, and/orstabilization processes that are conducted in cryopreservation kits.Different types of media may be used in these processes, for example,enzyme media, tumor digest media and/or cryopreservation media which mayallow for a controlled rate of freezing. In some embodiments,cryopreservation kit and/or components thereof may include indicatorsthat may be readable by an automated device. The device may then executea specific fully automatic method for processing tissue when inserted tosuch a device. The invention is particularly useful in a sampleprocessing, particularly automated processing.

In some instances, the cryopreservation kit and/or components thereofdescribed herein may be single use. Cryopreservation kits and/orcomponents thereof may be used in an automated and/or a semi-automatedprocess for the disaggregation, enrichment, and/or stabilization ofcells or cell aggregates. In some embodiments, bags for use in acryopreservation kit such as a collection bag may in some embodiments beused for multiple processes. For example, collection bags may berepeatedly sealed in different locations to create separate compartmentsfor processing of a tissue sample such as a biopsy sample and/or solidtissue.

Further, marks may be placed at various locations on bags, such astissue collection bags to indicate where the bags may be sealed,clamped, and/or affixed to an object. In some embodiments, marks showingwhere a bag may be clamped, sealed, and/or affixed to an object, such asinstrument, may be positioned on the bag prior to use. For example, oneor more marks may be positioned on a bag during manufacturing.

Seals may be formed during use with energy, for example, heat to createa weld zone. Seals formed during use may behave a width in a range fromabout 2.5 mm to about 7.5 mm. Generally, seal 140 is formed after tissuematerial is placed in bag 140 and may have a width of about 5 mm.

Seals may be tested for strength using a seal peel test (i.e., ASTMF88/F88M), and/or a burst test (i.e., ASTM F1140/F1140M or ASTMF2051/F2054M).

In some embodiments, a bag or a flexible container may withstand a forceof 100 Newtons during use when properly sealed and further secured witha clamp when positioned within a device for treatment and/or processing.A bag or a flexible container embodiment may be constructed to withstanda force of 75 Newtons during use when properly sealed and furthersecured with a clamp when positioned within a device for treatmentand/or processing.

When forming seals or welds on a flexible container such as a bag, forexample, a collection bag and/or a cryopreservation bag, a sealingdevice may be used to apply heat and/or pressure at a predeterminedtemperature, pressure, and amount of time depending on the material usedin the bag. For example, some heat sealers may require application ofheat and pressure for about eight seconds. After 8 seconds, heat may beturned off on the device, however, pressure may be applied for anadditional 2 to 3 seconds.

FIG. 12D shows a perspective view of an embodiment of a tissuecollection bag for sealing tissue therein for processing of theinvention. Indicators 27, 28 are positioned on bag 22 such that a usercan easily identify a patient during use. Further, these indicators maybe used to identify materials in the bags as well as track the progressduring a particular method of treatment for the materials in the bags.In some embodiments, a bag holds a volume of media in a range from about0.1 ml to about 25 ml and a volume of tissue in a range from about 0.1ml to about 10 ml in the bag during treatment. A ratio volume of mediato a volume of tissue in a bag during treatment should be in a rangefrom about 1.0 to about 2.5. In some embodiments, a ratio of the volumeof media to a volume of tissue is in a range from about 1.7 to about2.3. In particular, a ratio of the volume of media to a volume of tissueis in a range from about 2.0 to about 2.2.

As shown in FIG. 12D, marks 29 are positioned proximate open end 26 ofbag 22. During use marks 29 may be positioned on a bag based on a methodused to treat a tissue sample and/or biopsy sample. Marks may be placedon a bag during use, for example, based on the processing method beingused or to be used and/or the equipment to be used. In some embodiments,marks may be positioned on a bag during manufacturing. For example,positioning of marks for the locations of sealing and/or clamping mayvary based on the processing method and/or volume of tissue to betreated.

FIG. 12E shows a perspective view of a tissue collection bag. Tissue maybe sealed in bag 22 processing. Connector 24 may provide access to thebag. As shown connector 24 may be connected to other devices such asfilter, bags, etc. using tubing 25. Ports 20 may be used to take samplesfrom bag 22 and/or provide materials from bag 22 during use.

FIG. 13A shows a front view of a bag used for tissue collection. Tissuemay be sealed within hag during use. Bag 30 may be manufactured havingsealed edge 31. As shown in FIG. 13A, sealed edges 31 may be located onthree edges and fourth edge may include open section 36.

Positioners 33 on bag 30 may be used to position a bag. For example, oneor more positioners may be used to ensure that bag can be treatedproperly during use, for example, positioning proximate an instrument.In some systems, the positioners may facilitate the use of the bagsdescribed herein in automated systems. In particular, positioners may beused to move bag through an automated system.

As shown in FIG. 13B, bag 30 may have indicators 36, 37 used to identifya sample, for example, an indicator that identifies a patient from whoma tissue sample or biopsy has been taken or obtained. Use of anindicator 37 such as a QR code may allow for tracking of process stepsfor a specific sample such that it is possible to follow the samplethrough a given process.

FIG. 13C shows a front view of a tissue collection hag. Tissue may besealed within a bag and treated and/or processed therein. Bag 30 mayhave indicators 37, 38 used to identify a sample, for example, anindicator that identifies a patient from whom a tissue sample or biopsyhas been taken or obtained. Use of indicator 37 such as a QR code mayallow for tracking of process steps for a specific sample such that itis possible to follow the sample through a given process. Positioners 33may be used to position bag 30 for treatment. Connector 34 may allowtissue, treated tissues, etc. to couple to other device through tubing35.

FIG. 13D depicts a front view of a tissue collection bag havingindicators 37, 38 used to identify a sample. Use of an indicator 37 suchas a QR code may allow for tracking of process steps for a specificsample such that it is possible to follow the sample through a givenprocess. Marks 39 and/or positioners 33 may be used to controlpositioning of the bag during processing and/or treatment. Marks placedproximate an open end to indicate where to position, seal and/or clampthe bag during use. Bag 30 may be manufactured having sealed edges 3 LAs shown in FIG. 13D, sealed edges 31 may be located on three edges andfourth edge may include open section 36.

FIG. 13E shows a front view of a tissue collection bag which is capableof being sealed after tissue is placed therein. Connectors 34 and ports32 may provide access to the bag. One or more ports may be positioned ona collection bag such that the ports allow for input of media and/orreagents and/or extraction of sample from the bags.

As shown connector 34 may be coupled to other devices such as filter,bags, etc. using tubing 35. Marks and indicators may be placed one ormore sides of the hag depending on use. In particular, as shown if FIG.13E, positioners 33, marks 39, and/or indicators 37, 38 may be used toposition bag 30 for processing such as applying agitation, sealing,e.g., by heat sealing (which may be part of the instrument forprocessing), addition of materials for processing and/or extraction.Advantageously, prior to application of processing, the collection bagis clamped or affixed into an instrument for processing and/or sealed,e.g., heat sealed.

FIG. 14 shows a back view a bag for tissue collection. In particular,bag 40 is capable of being sealed with tissue positioned therein andprocessed. Seal may be positioned proximate open end 46 andsubstantially parallel thereto. As shown connector 44 may be connectedto other devices such as filter, bags, etc. using tubing 46. Bag 40 maybe manufactured having sealed edge 41. As shown in FIG. 14 , sealededges 41 may be located on three edges and fourth edge may include opensection 46. Positioners 43 may be surrounded by manufactured sealed edge41.

FIG. 15 depicts a side view of bag 50 for use in tissue collectioncapable of sealing tissue therein and allowing processing of the tissueduring use of the bag. Bag 50 may be coupled to tubing 54 by connector52.

FIG. 16A shows a top view of an unsealed tissue collection bag. Bag 60may include sealed portions 66 and open portion 64. Connector 62 isvisible through bag 60. After placing tissue in bag open portion of topof bag 60 may be sealed.

FIG. 16B shows a bottom view of the tissue collection bag 60 havingsealed edges 66 for sealing tissue therein for processing. Connector 62visible on bag 60.

FIG. 17A shows a top view of partially open bag. Bag 70 may includesealed portions 76 and open portion 74. Connector 72 is visible throughbag 70. After placing tissue in bag open portion of top of bag 70 may besealed.

FIG. 17B shows a bottom view of the tissue collection bag for sealingtissue therein for processing. Connector 72 is visible on bag 70.

FIG. 18A depicts a top view of a partially open bag. Tissue may beinserted through open end 84 of bag 80. Connector 82 is shown positionedat the bottom of bag 80.

FIG. 18B shows a top view of a fully open bag for the collection and/orprocessing of tissue. Open end 84 of bag 80 may receive tissue forprocessing such as treatment, isolation, and/or separation. Sealed edges86 may be created during manufacturing.

FIG. 19A depicts a top view of partially open hag 90 having sealed edges96 on the sides of the bag. As shown, tissue may be inserted throughopen end 94 of bag 90. Connector 92 is shown positioned at the bottom ofbag 90.

FIG. 19B shows a top view of a fully open bag for the collection and/orprocessing of tissue having sealed edges 96 on the sides of the bag.Open end 94 of bag 90 may receive tissue for processing such astreatment, isolation, and/or separation. Connector 92 is shownpositioned at the bottom of bag 94.

FIGS. 20A-20E show a front view of embodiments of tissue collectionbags. As shown in FIG. 20A, bag 100 having sealed edges 101 and open end102 may be connected to devices (not pictured) via tubing 105 and/orconnectors 104. For example, connector 104 is positioned in bag 100while y-connectors 10 ⁶ may be positioned along tubing. FIG. 20B shows afurther embodiment of hag 100 including indicators 107, 108 such that auser can identify a patient from whom a tissue sample or biopsy has beentaken or obtained.

In addition, an embodiment of bag 100 that includes mark 109 andindicators 107, 108 is depicted in FIG. 20C. Use of positioners 103 mayallow for consistent positioning of bags that allow for consistentprocessing of tissue within bags. Indicators 107, 108 identify sampleswith either sample and/or patient information. In some instances,indicators may be used to identify and/or track a sample, such as atissue sample and/or biopsy sample. FIG. 20D depicts bag 100 havingmultiple indicators 107, 108 and marks 109. Marks may show locationswhere bag 100 is to be sealed. For example, marks 109 may indicatelocations where bag 100 should be sealed, clamped, and/or couple toanother device. Marks for sealing may be positioned proximate an openedge of the bag, for example, such marks may be positioned apredetermined distance from the open edge. Marks for sealing may besubstantially parallel to the open edge in some embodiments. As shownbag 100 may include connector 104 and tubing 105.

In an embodiment as shown in FIG. 20E, bag 100 includes ports 110 andconnector 104. Ports may allow for addition of materials and/or removalof material from the sample. For example, during processing of thetissue, samples may be taken at multiple times throughout processing.Further, ports 110 may allow aseptic input of media and/or reagents intobag 100.

FIG. 21A shows a front view of bag 100 for the collection and/orprocessing of tissue. Tissue may be placed in bag 100 through open end102. Connector 104 may be used to couple bag 100 with tubing 105, andclamp 112.

FIGS. 21B-21E show front views of additional embodiments of hag 100.FIGS. 21B-11D show various configurations including indicators 107, 108and/or marks 109. Bags may include indicators such as codes, letters,words, names, alphanumeric codes, numbers, images, bar codes, quickresponse (QR) codes, tags, trackers such as smart tracker tags orBluetooth trackers, and/or any indicator known in the art. In someembodiments, indicators may be printed on, etched on, and/or adhered toa surface of a component of a kit. Indicators may also be positioned ona bag using an adhesive, for example, a sticker or tracker may be placedon a bag and/or on multiple bags. Collection bags and/orcryopreservation kit may include multiple indicators such as numericcodes and/or QR codes.

Indicators, for example QR codes, tags such as smart tags, and/ortrackers may be used to identify a sample within a bag as well as toinstruct a device's processor such that the device runs a specificprogram according to a type of disaggregation, enrichment, and/orstabilization processes that are conducted in cryopreservation kits.

FIG. 21E depicts a front view of another embodiment of bag 100 used forcollection, processing, treatment, and/or isolation of materials. Tissueto be treated may be sealed within bag 100. Tubing 105 may couple bag100 through connector 104 to clamp 112. Ports 114 may allow for inputand/or removal from bag 100. For example, ports may allow for samplingand/or allow for aseptic input of media and/or reagents into a flexiblecontainer, such as a bag of the cryopreservation kit.

FIG. 22A shows a front view of another embodiment of a tissue collectionbag 120 having sealed edge 121 for sealing tissue therein forprocessing. Bag 120 includes positioner 123 and connector 124 coupled totubing 125.

FIG. 22B shows a front view of tissue collection bag 120 having sealededges 121 and open end 122. Indicators 127, 128 may be positioned on bag120 such that they can be easily accessed by an automated system.Openings defining positioners 123 may be surrounded by sealed edges 121.Indicators may be used to identify the patient from whom a tissue sampleor biopsy has been taken or obtained.

As shown in FIG. 22C, bag 120 includes indicators 127, 128 and mark 129.FIG. 22D depicts shows a collection bag 120 having multiple marks 129.Marks for sealing may be positioned proximate an open edge of the bag.Such marks may be positioned a predetermined distance from the openedge. Marks for sealing may be substantially parallel to the open edgein some embodiments.

FIG. 23 depicts a front view of sealed bag 130 positioned such that thebottom of bag 130 is shown at the top of the page with tubing 135emerging from connector 134. Bag 130 includes indicator 137 on sealedportion 131 of bag 130. An indicator on the sealed portion may bepositioned during and/or after sealing of bag 130. Generally, the bag issealed after tissue is provided. Indicator 138 on a surface of bag 130may be a bar code. Positioners 133 may be positioned proximate connector134.

Bags, such as collection bags and/or cryopreservation bags, and anyassociated tubing may be generally clear, transparent, translucent, anycolor desired, or a combination thereof. Tissue collection bags and/ortubing may be generally fabricated in ways analogous to the fabricationof closed and/or sealed blood and/or cryopreservation bags and theassociated tubing. Tubing in the invention may be constructed from anydesired material including, but not limited to polyvinyl chloride (PVC).For example, PVC may be a desired material as PVC is advantageous forwelding and/or sealing.

A collection bag, such as a tissue collection bag of the invention mayinclude at least a portion of the bag for receiving tissue made from apredetermined material such as a polyolefin polymer, ethylene vinylacetate (EVA), copolymers such as vinyl acetate and polyolefin polymerblend (i.e., OriGen Biomedical EVO film), and/or a material includingEVA. Materials for use in the bag may be selected for a specificproperty and/or a selection of properties, for example, salability suchas heat sealability, gas permeability, flexibility for example lowtemperature flexibility, elasticity for example low temperatureelasticity, chemical resistance, optical clarity, biocompatibility suchas cytotoxicity, hemolytic activity, resistance to leaching, having lowparticulate.

As shown in FIG. 24 , bag 140 may include multiple marks 141, 142 thatare placed such that if the areas including marks are sealed,compartments 143 may be formed in bag 140. Bag 140 has pre-weldedsections 145 that are formed during manufacture of the bag that may beused in the formation of the compartments for samples during use. FIG.24 depicts an embodiment of a collection bag that is capable of beingformed such that it has multiple compartments. Each compartment may beformed in a bag by placement of multiple seals and/or welds (e.g., heatsealed). For example, after placing a tumor suspension in a collectionhag the open end may be welded shut and additional marks 141 such asweld lines 142 may be welded using energy such as heat to formcompartments.

Positioners 143 on bag 140 ensure that the bag is positioned correctlywith respect to instruments, such as sealing devices like RF heatsealers and/or injectors.

Seals may be formed during use with energy, for example, heat to createa weld zone. Seals formed during use may behave a width in a range fromabout 2.5 mm to about 7.5 mm. Generally, seal 140 is formed after tissuematerial is placed in bag 140 and may have a width of about 5 mm.

Seals may be tested for strength using a seal peel test (i.e., ASTMF88/F88M), and/or a burst test (i.e., ASTM F1140/F1140M or ASTMF2051/F2054M).

In some embodiments, a hag or a flexible container may withstand a forceof 100 Newtons during use when properly sealed and further secured witha clamp when positioned within a device for treatment and/or processing.A bag or a flexible container embodiment may be constructed to withstanda force of 75 Newtons during use when properly sealed and furthersecured with a clamp when positioned within a device for treatmentand/or processing.

When forming seals or welds on a flexible container such as a bag, forexample, a collection bag and/or a cryopreservation bag, a sealingdevice may be used to apply heat and/or pressure at a predeterminedtemperature, pressure, and amount of time depending on the material usedin the bag. For example, some heat sealers may require application ofheat and pressure for about eight seconds. After 8 seconds, heat may beturned off on the device, however, pressure may be applied for anadditional 2 to 3 seconds.

In some systems, the positioners may facilitate the use of the bagsdescribed herein in automated systems. Thus, tissues that have beenplaced in bag 140 may be split into separate compartments 144, 146, 147.As shown, each compartment 144, 146, 147 includes ports 148, 149, 150,respectively. Each port may allow for direct access into compartments.This may allow for individualized additions, banking, and/or testing ofsamples. For example, a sealed collection bag may facilitate banking andtesting of TIL for suitability and/or microbiological properties ofcomplex samples. As this type of testing may require a small aliquot ofthe digested material to be frozen in the collection bag such that thesmall aliquot of the digested material can be thawed separately. In someembodiments, hag 140 as depicted in FIG. 24 may be used as a collectionhag and/or a cryopreservation bag.

FIG. 25 shows a front view of an embodiment of a collection bag. In thisembodiment, collection bag 152 has a length of about 150 mm (i.e., 15cm) and a width of about 90 mm (i.e., 9 cm). Bag 152 includes openingsacting as positioners 160. One or more positioners may be used tocontrol the orientation of the bag to ensure that the bag is positionedproperly for processing and/or treatment during use, for example,positioning proximate an instrument. In some systems, the positionersmay facilitate the use of the bags described herein in automatedsystems. In particular, positioners may be used to move bag through anautomated system. Seal 156 is about 5 mm. Seals may be formed during useusing energy, for example, heat to create a weld zone. Seals may have awidth in a range from about 2.5 mm to about 7.5 mm. Generally, seal 156is formed after tissue material is placed in hag 152. As shown in FIG.25 , hag 152 has pre-welded sections 158 that are formed duringmanufacture of the bag.

As shown in FIG. 26 , a collection bag may be coupled to tubing and avalve. In some embodiments, bags may have a length in a range from about10 cm to about 50 cm. In particular, bags for use in the inventiondescribed herein may have a length in a range from about 15 cm to about30 cm. For example, bags may have a length in a range from about 18 cmto about 22 cm. Bag 162 as shown in FIG. 26 has a length of about 20 cm.Collection bags for use as described herein may have a width in a rangefrom about 6.8 cm to about 8.8 cm. As shown in FIG. 26 , collection bag162 has a width of about 7.8 cm. Valves including, but not limited toneedle free valves may be used at points along the tubing. For example,needle free valve 164 is positioned approximately 20 cm from bag 162coupled by tubing 166. Tubing 166 extends from needle free valve 164 forat least 10 cm before another element or component is added.

As depicted in FIG. 27A, open bag 170 is coupled to tubing 172, 174, 176prior to use. Bag 170 may be constructed from a sealable material. Inparticular, the bags may be sealable using a heat sealer such as, forexample, a benchtop heat-sealing device. Some of the tubing, for exampletubing 174 may be non-weldable. Valves including but not limited toneedle free valves may be used at points along the tubing. For example,needle free valves 178 are positioned at ends of tubing 174, 176.

In some embodiments, bags may have a length in a range from about 10 cmto about 50 cm. In particular, bags for use in the invention describedherein may have a length in a range from about 15 cm to about 30 cm. Forexample, bags may have a length in a range from about 18 cm to about 22cm. Bag 170 as shown in FIG. 27A has a length of about 20 cm.

FIG. 27B shows a front view of an embodiment of a collection bag thathas been sealed, for example, after deposition of material within thebag. Bag 180 is constructed from a sealable material. In particular, thebags may be sealable using a heat sealer such as, for example, abenchtop heat-sealing device. Seals may be positioned proximate an openedge of the bag, in some instances, marks may be positioned apredetermined distance from the open edge. Seals may be substantiallyparallel to the open edge in some embodiments.

Some of the tubing, for example tubing 182, 184, 186 may be weldable.Weldable tubing may be made from a polymer material, for example,polyvinyl chloride (PVC).

Valves including, but not limited to needle free valves may be used atpoints along the tubing. For example, needle free valves 188 arepositioned at ends of tubing 184, 186. In some embodiments, bags mayhave a length in a range from about 10 cm to about 40 cm. In particular,bags for use in the invention described herein may have a length in arange from about 15 cm to about 30 cm. For example, bags may have alength in a range from about 18 cm to about 22 cm. Bag 180 as shown inFIG. 27A has a length of about 20 cm.

As shown in FIG. 28 , an embodiment of a cryopreservation kit is shownfacing upwards and includes open bag 190 and a cryopreservation bag 192.As shown cryopreservation bag 192 may include indicators 193, 194.Cryopreservation bags may need to be suitable for cryopreservation witha cryoprotectant such as dimethyl sulfoxide (“DMSO”). In someembodiments, cryopreservation bags may be constructed so that the bagsmay hold a volume of material in a range from about 5 ml to about 45 ml.In particular, a cryopreservation bag may include accommodate a volumeof material in a range from about 10 ml to about 35 ml. For example,some embodiments include cryopreservation bags that may accommodate avolume of material to be stored in a range from about 15 ml to about 30ml. Cryopreservation bag 192 may have sized such that a desiredpredetermined volume is achieved. In some embodiments, acryopreservation bag may have a width in a range from about 4 cm toabout 11 cm and a length in a range from about 10 cm to about 18 cm. Forexample, a cryopreservation bag may have a width in a range from about5.8 cm to about 9.8 cm and a length in a range from about 12 cm to about16 cm. In particular, an embodiment of a cryopreservation bag asdepicted in FIG. 28 may have a width of about 7.8 cm and length of about14 cm.

Prior to use the cryopreservation kit and/or specific components thereofmay be sterilized. For example, bags 190, 192 may be sterilized.Materials used to form bags 190, 192 may be heat sealable. Materials foruse in the bags may include, but is not limited to polymers such as EVA,polyamides (e.g., nylons), and combinations thereof. Open bag 190 may beused for processing and/or disaggregation after closing the bag using aseal and/or a clamp (not shown).

Kit 191 further includes valves 195, 196, clamps 197, 198, tubing 199,and filter 200. Filter 200 may be an inline filter, a blood filter, suchas a blood administration filter, a biological filter, and/or an in-lineclump removal filter. The filter may be configured to remove materialsfrom the processed tissue above a predetermined size to form a desiredmaterial. For example, lumps of tissue may be separated from thedisaggregated tissue using the filter. In particular, a tissuecomposition entering tubing after being filtered may have constituentshaving an average size of less than about 200 μm such that a desiredmaterial is formed. For example, the desired material may include TILs(tumor infiltrating lymphocytes) having an average size of less thanabout 170 pm.

A filter may be selected such that the processed tissue compositionentering from tubing may be enriched such that after the filter thedesired material flows into tubing in the direction of the stabilizationelement having constituents having a size in a range from about 15 pm toabout 500 pm. In some embodiments, a filter may be configured such thata tissue composition entering tubing in the direction of thestabilization element after being filtered has constituents having asize in a range from about 50 pm to about 300 IJM. For example, a filtermay, in an embodiment, be configured such that a tissue compositionentering tubing after being filtered has constituents having a size in arange from about 150 pm to about 200 pm.

In some embodiments, a filter of the enrichment element may removematerials from the processed tissue outside of a predetermined sizerange from about 5 pm to about 200 pm to form a desired material. Forexample, the desired material may include TILs (tumor infiltratinglymphocytes) having an average size in a range from about 5 pm to about200 pm. Valves 195, 196 may be placed a predetermined distance from acollection bag. For example, needle free valve 195 may be positionedabout 20 cm from collection bag 190. Valves such as needle free valvesmay be used to add materials to collection bag 190. For example, enzymemedia may be inserted into needle free valve 195 in order to add themedia to collection bag 190.

In some embodiments, after such a valve there may be a predeterminedamount of tubing to allow space to weld on additional components for thecryopreservation kit. For example, after some valves at least ten (10)cm of tubing may be positioned before next element. Tubing 199 may besealable and/or weldable. For example, materials for tubing may include,but is not limited to PVC (polyvinyl chloride), and/or other materialsknown in the art. In some embodiments, tubing may be sized to fitconnectors. For example, tubing may have an inner diameter in a rangefrom about 1.5 mm to about 4.5 mm and an outer diameter in a range fromabout 2.1 mm to about 6.1 mm. For example, an embodiment of acryopreservation kit may include tubing having an inner diameter in arange from about 2.9 mm to about 3.1 mm and having an outer diameter ina range from about 4.0 mm to about 4.2 mm. Tubing used incryopreservation kit 191 may vary in length with individual tubingelements having a length in a range from about 1 cm to about 30 cm. Forexample, as depicted in FIG. 28 lengths of individual tubing elementsmay vary from about 5 cm to about 20 cm.

Clamps 197, 198 as depicted in FIG. 28 may be used to inhibit and/orprevent movement of enzyme media and/or digested tissue into the filter.For example, clamp 197 may be used to inhibit and/or prevent movement ofenzyme media and/or digested tissue into the filter prior to a desiredfiltration step. Clamp 198 may inhibit and/or prevent undesired movementof the cryoprotective agent into the filter.

FIG. 29 shows a top view of an embodiment of a cryopreservation kitsimilar to the kit 191 shown in FIG. 28 , however kit 201 is facingdownwards. FIG. 29 depicts a position at which collection bag 202 may beclosed.

FIG. 30 shows a top view of an embodiment of a cryopreservation kitfacing upwards including closed collection bag 206 and cryopreservationbag 208. In some embodiments, cryopreservation bag 208 may include ports215, 216 that allow for sampling, permit aseptic input of media and/orreagents into the cryopreservation bag. Cryopreservation kit 205 mayinclude filter 214, valves 209, 210, clamps 211, 212 and tubing 222.

Filter 214 may be an inline filter, a biological filter, a blood filtersuch as a blood administration filter and/or an in-line clump removalfilter. The filter may be configured to remove materials above apredetermined size. For example, lumps of tissue may be separated fromthe disaggregated tissue using the filter. A filter may be selected suchthat tissue composition entering tubing after the filter may haveconstituents having a size in a range from about 15 pm to about 500 μm.In some embodiments, a filter may be configured such that a tissuecomposition entering tubing after being filtered has constituents havinga size in a range from about 50 pm to about 300 pm. For example, afilter may, in an embodiment, be configured such that a tissuecomposition entering tubing after being filtered has constituents havingan average size in a range from about 150 pm to about 200 pm. Inparticular, a tissue composition entering tubing after being filteredmay have constituents having an average size of less than about 170 μm.

Valves 209, 210 may be placed a predetermined distance from a collectionbag. For example, needle free valve 209 may be positioned about 20 cmfrom collection bag 206. Valves such as needle free valves may be usedto add materials to collection bag 206. For example, enzyme media may beinserted into needle free valve 209 in order to add the media tocollection bag 206.

In some embodiments, after such a valve there may be a predeterminedamount of tubing to allow space to weld on additional components for thecryopreservation kit. For example, after some valves at least ten (10)cm of tubing may be positioned before next element. Tubing 222 may besealable and/or weldable. For example, materials for tubing may include,but is not limited to PVC and/or other materials known in the art. Insome embodiments, tubing may be sized to fit connectors. For example,tubing may have an inner diameter in a range from about 1.5 mm to about4.5 mm and an outer diameter in a range from about 2.1 mm to about 6.1mm. For example, an embodiment of a cryopreservation kit may includetubing having an inner diameter in a range from about 2.9 mm to about3.1 mm and having an outer diameter in a range from about 4.0 mm toabout 4.2 mm. Tubing used in cryopreservation kit 205 may vary in lengthwith individual tubing elements having a length in a range from about 1cm to about 30 cm. For example, as depicted in FIG. 30 lengths ofindividual tubing elements may vary from about 5 cm to about 20 cm.

Clamp 211, 212 as depicted in FIG. 30 may be used to inhibit and/orprevent movement of enzyme media and/or digested tissue into the filter.For example, clamp 211 may be used to inhibit and/or prevent movement ofmedia enzyme solution and/or digested tissue into the filter prior to adesired filtration step. Clamp 212 may inhibit and/or prevent undesiredmovement of the cryoprotective agent into the filter.

FIG. 31 shows a side view of an embodiment of a cryopreservation kitfacing upwards that includes closed collection bag 226 andcryopreservation bag 228. Cryopreservation bag 228 may include port 242.Port 242 provides access to cryopreservation hag 228. Valves 232, 238and clamps 234, 236 may be positioned around filter 230 and used tocontrol movement of the fluid within the cryopreservation kit 224.

FIG. 32 shows an end view of an embodiment of a cryopreservation kit.Sealed bag 226 and filter 230 are visible. Sealed bag 226 may be coupledto filter 230 using tubing, valves, and/or clamps.

FIG. 33 shows a top view of an embodiment of a collection bag. Bag 232is shown as open and includes indicators 234, 236 and marks 238, 240.Marks may be used to show where portions of a bag should be sealedand/or clamped. Marks for sealing may be positioned proximate an openedge of the bag. Such marks may be positioned a predetermined distancefrom the open edge. Marks for sealing may be substantially parallel tothe open edge in some embodiments.

Bag 232 includes positioners 244 and connector 246. Connector 246couples hag 232 to tubing 248. Connecter 246 may allow tubing 248 tosplit into tubing 250, 252 that include clamps 254, 256 and/or ports258, 260.

FIG. 34 shows a front view of an embodiment of a cryopreservation kitthat includes a collection bag 264, clamps 266, 268, filter 270, tubing272, ports 274, 276, valves 278, connector 280, and cryopreservation bag282. The collection bag and the associated tubing may be formed using atleast some EVA material. In some embodiments, the collection bag and/ortubing may be formed from EVA. Clamps 266, 268 may be pinch clamps.Connector 280 is a four-way connector and may be used to couple tubingfrom filter 270 to valves 278, for example needle free valves, as wellas to tubing coupled to cryopreservation bag 282.

FIG. 35 shows a front view of an embodiment of a cryopreservation kitthat includes collection bag 284, ports 286, clamps 288, 296, valves290, 292, filter 298, and cryopreservation bag 294. As depicted, valves290, 292 may be needle free valves capable of receiving materials foruse in the kit during processing. For example, materials to be providedvia valves 290, 292 include, for example, tumor digest media and/or acryoprotectant or cryopreservation media such as dimethyl sulfoxide(“DMSO”) and/or solutions thereof, such as 55% DMSO and 5% Dextrancryopreservation media (e.g., BloodStor 55-5). Syringes 300, 302 may beused to provide tumor digest media and a 55% DMSO solution, such as 55%DMSO and 5% Dextran cryopreservation media, respectively, through needlefree valves 290, 292. During processing materials may be selectivelyprovided to the cryopreservation kit at predetermined times. Further,clamps may be used to control the flow of provided materials such astumor digest media and/or a cryoprotectant, such as a DMSO solution maybe provided to the devices such as the collection bag, the filter,and/or the cryopreservation bag at predetermined times.

FIG. 36A shows a front view of an embodiment of a cryopreservation kitthat is capable of being secured in a device such as a digestor. Asshown collection bag 304 is enclosed at least partially by bracket 306during use. Bracket may position collection bag 304 such that processingcan occur in an efficient manner. FIG. 36A depicts collection bag 304that has weld 310 and utilizes clamp 312 proximate weld 310 during useto reduce pressure on weld 310. Tissue introduced during use may bedistributed substantially evenly in collection bag 304 such that tissuemay be treated using paddles 314, 316 from a device. Cryopreservationbag 330 has multiple sections 332 each having their own port 334.

A side view of an embodiment of a collection bag secured using a bracketis depicted in FIG. 36B. Bracket 336 may be used to secure a collectingbag. Bracket 336 includes hinge 338, top side 340, bottom side 342,clamp 344, protrusion 346 and latch 348. During use clamp 344 may bepositioned proximate a weld on collection bag (FIG. 36A). Protrusion 346on bracket 336 is constructed such that it would be positioned proximatea surface of the collection bag and protrude up into collection bagduring use. In some embodiments, protrusion 346 may reduce and/orinhibit movement of tissue and/or media during use to ensure thatprocessing of tissue is substantially similar along the length of thecollection bag. For example, the protrusion may be constructed such thatit reduces and/or inhibits sliding of tissues between paddles (shown inFIG. 36A). Bracket 336 may also include latch 348 to ensure thatcollection bag is secured.

FIG. 36C shows an exploded view of clamp 344 including ridges 350 foruse with a collection bag. In particular, during use clamp 344 may bepositioned proximate a weld on a collection bag to reduce the risk ofweld and/or seal failures.

FIG. 37 shows a top view of an embodiment of a cryopreservation kit thatincludes collection bag 354, filter 356, valves 362, 364, clamps 358,360, tubing 368, and cryopreservation bag 366. Tubing length betweenvarious components of the cryopreservation kit 352 may vary.

FIG. 38 shows a view of an embodiment of a cryopreservation kitpositioned face down that includes collection bag 354, filter 356,valves 362, 364, clamps 358, 360, tubing 368, and cryopreservation bag366.

Two or more bags may be coupled together to ensure that disaggregatedproduct material may be properly stored in a particular embodiment.

In some embodiments, the invention may include an automated device forsemi-automated aseptic disaggregation, enrichment, and/or stabilizationof cells and/or cell aggregates from tissue, for example a solidmammalian tissue. An automated device for use with the invention mayinclude a programmable processor and a cryopreservation kit. In someembodiments, the cryopreservation kit may be single use. aseptic kit.The invention further relates to a semi-automatic aseptic tissueprocessing method.

In some embodiments, bags such as a collection bag may be used in acollection kit. Bags have an open end allowing for the addition of asample, such as a tissue sample. A connector may couple the bag totubing in a collection kit. Tubing material may be sealable and/orweldable. For example, the tubing may be sealed using energy such asheat, radio frequency, etc. The tubing material may be made from PV A.

In some embodiments, tubing may be coupled to a valve to allow additionof one or more media enzyme solutions including, but not limited tocollagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, LiberaseHI, pepsin, or mixtures thereof. For example, the valve may be a needlefree valve.

Tubing used in the cryopreservation kit may include tubing having anouter diameter in a range from about 3.0 mm to about 5.0 mm with aninner diameter of the tubing in a range from about 2.0 mm to about 4 mm.In particular, tubing may have an outer diameter of 4.1+/−0.1 mm and aninner diameter of about 3.0+/−0.1 mm. The length of tubing may depend onthe configuration of the collection kit. For example, an embodiment of acollection kit may include tubing having a length in a range from about10 cm to about 20 cm.

In some embodiments of the collection kit prototype may include one ormore clamps to inhibit and/or prevent movement of tissue and/or enzymemedia. In particular, enzyme media and/or tissue may be inhibited frommoving into a filter before a filtration step The invention is furtherdescribed by the following numbered paragraphs:

1. A single use aseptic kit comprising: a disaggregation module forreceipt and processing of material comprising solid mammalian tissue; anoptional enrichment module for filtration of disaggregated solid tissuematerial and segregation of non-disaggregated tissue and filtrate; and astabilization module for optionally further processing and/or storingdisaggregated product material, wherein each of said modules comprisesone or more flexible containers connected by one or more conduitsadapted to enable flow of the tissue material there between; and whereineach of said modules comprises one or more ports to permit aseptic inputof media and/or reagents into the one or more flexible containers.

2. The single use aseptic kit of paragraph 1, wherein the one or moreflexible containers comprise a resilient deformable material.

3. The single use aseptic kit of paragraph 1 or 2, wherein the one ormore flexible containers of the disaggregation module comprises one ormore sealable openings.

4. The single use aseptic kit of paragraph 3, wherein the flexiblecontainer of the disaggregation module comprises a heat sealable weld.

5. The single use aseptic kit of any preceding paragraph, wherein theone or more flexible containers comprises internally rounded edges.

6. The single use aseptic kit of any preceding paragraph, wherein theone or more flexible containers of the disaggregation module comprisesdisaggregation surfaces adapted to mechanically crush and shear thesolid tissue therein.

7. The single use aseptic kit of any preceding paragraph, wherein theone or more flexible containers of the enrichment module comprisesfilter which retains a retentate of cellularized disaggregated solidtissue.

8. The single use aseptic kit of any preceding paragraph, wherein theone or more flexible containers of the stabilization module comprisesmedia formulation for storage of viable cells in solution or in acryopreserved state.

9. The single use aseptic kit of any preceding paragraph, wherein thekit further comprises a digital, electronic or electromagnetic tagindicator.

10. The single use aseptic kit of paragraph 9, wherein the tag indicatorrelates to a specific a program that defines: a type of disaggregationand/or enrichment and/or stabilization process; one or more types ofmedia used in those processes; including an optional freezing solutionsuitable for controlled rate freezing.

11. The single use aseptic kit of any preceding paragraph, wherein thesame flexible container can form part of one or more disaggregationmodule, the stabilization module and the optional enrichment modules.

12. The single use aseptic kit of any preceding paragraph, wherein thedisaggregation module comprises a first flexible container for receiptof the tissue to be processed.

13. The single use aseptic kit of any preceding paragraph, wherein thedisaggregation module comprises a second flexible container comprisingthe media for disaggregation.

14. The single use aseptic kit of any preceding paragraph, wherein theoptional enrichment module comprises the first flexible container and athird flexible container for receiving the enriched filtrate.

15. The single use aseptic kit of any preceding paragraph, wherein boththe disaggregation module and the stabilization module comprise thesecond flexible container and wherein the second container comprisesdigestion media and stabilization media.

16. The single use aseptic kit of any preceding paragraph, wherein thestabilization module comprises a fourth flexible container comprisingstabilization media.

17. The single use aseptic kit of any preceding paragraph, wherein thestabilization module also comprises the first flexible container and/orthird flexible container for storing and/or undergoing cryopreservation.

18. Use of the single use aseptic kit according to any precedingparagraph in a semi-automated process for the aseptic disaggregation,stabilization and optional enrichment of mammalian cells or cellaggregates.

19. An automated device for semi-automated aseptic disaggregation and/orenrichment and/or stabilization of cells or cell aggregates frommammalian solid tissue comprising: a programmable processor; and thesingle use aseptic kit of any of paragraphs 1 to 17.

20. The automated device of paragraph 19, further comprising radiofrequency identification tag reader to recognize the single use kit.

2 L The automated device of paragraph 19 or 20, wherein the programmableprocessor is capable of recognizing the single use aseptic kit via thetag and subsequently executes the kit program defining the type ofdisaggregation, enrichment and stabilization processes and therespective media types required for those processes.

22. The automated device of any preceding paragraph, wherein theprogrammable processor is adapted to communicate with and control one ormore of: the disaggregation module; the enrichment module; and thestabilization module.

23. The automated device of paragraph 22, wherein the programmableprocessor controls the disaggregation module to enable a physical and/orbiological breakdown of the solid tissue material.

24. The automated device of paragraph 23, wherein the programmableprocessor controls the disaggregation module to enable a physical andenzymatic breakdown of the solid tissue material.

25. The automated device of paragraph 24, wherein the enzymaticbreakdown of the solid tissue material is by one or more media enzymesolutions selected from collagenase, trypsin, lipase, hyaluronidase,deoxyribonuclease, Liberase HI, pepsin, or mixtures thereof.

26. The automated device of any one of paragraphs 19-25, wherein theprogrammable processor controls disaggregation surfaces within thedisaggregation flexible containers which mechanically crush and shearthe solid tissue, optionally wherein the disaggregation surfaces aremechanical pistons.

27. The automated device of any one of paragraphs 19-25, wherein theprogrammable processor controls the stabilization module to cryopreservethe enriched disaggregated solid tissue in the container, optionallyusing a programmable temperature.

28. The automated device of any preceding paragraph wherein the devicefurther comprises one or more of the additional components in anycombination: sensors capable of recognizing whether a disaggregationprocess has been completed in the disaggregation module prior totransfer of the disaggregated solid tissue to the optional enrichmentmodule; weight sensors to determine an amount of media required in thecontainers of one or more of the disaggregation module; the enrichmentmodule; and/or the stabilization module and control the transfer ofmaterial between respective containers; sensors to control temperaturewithin the containers of the one or more of the disaggregation module;the enrichment module; and/or the stabilization module; at least onebubble sensor to control the transfer of media between the input andoutput ports of each container in the module; at least one pump,optionally a peristaltic pump, to control the transfer of media betweenthe input and output ports; pressure sensors to assess the pressurewithin the enrichment module; one or more valves to control a tangentialflow filtration process within the enrichment module; and/or one or moreclamps to control the transfer of media between the input and outputports of each module.

29. The automated device of any preceding paragraph, wherein theprogrammable processor is adapted to maintain an optimal storagetemperature range in the stabilization module until the container isremoved; or executes a controlled freezing step.

30. The automated device of any preceding paragraph, further comprisinga user interface.

3L The automated device of paragraph 23, wherein the interface comprisesa display screen to display instructions that guide a user to inputparameters, confirm pre-programmed steps, warn of errors, orcombinations thereof.

32. The automated device of any preceding paragraph, wherein theautomated device is adapted to be transportable.

33. A semi-automatic aseptic tissue processing method comprising:automatically determining aseptic disaggregation tissue processing stepsand their associated conditions from a digital, electronic orelectromagnetic tag indicator associated with the aseptic processingkit, optionally in accordance with the kit according to any ofparagraphs 1 to 17; placing a tissue sample into a flexible plasticcontainer of the disaggregation module of the aseptic processing kit;and processing the tissue sample by automatically executing the one ormore tissue processing steps by communicating with and controlling thedisaggregation module; the optional enrichment module; and thestabilization module.

Procedures for Collection of Tumor Material, Cryopreseration, and TILManufacure

The starting material for TIL manufacturing is a disaggregated andcryopreserved cell suspension containing autologous TIL and tumor cellsfrom an eligible patient. An exemplary flow diagram is provided (FIG. 65) for collection and processing of the tumor starting material.

The tumor is surgically resected and then trimmed to remove visiblynecrotic tissue, visibly healthy (non-cancerous) tissue, fat tissue, andexcess blood. The trimmed tumor weight should be greater than or equalto 2 grams (≥2 grams). Tumors weighing over 7 g may be divided intosmaller portions and individually disaggregated.

Each tumor fragment is placed into an individual sterile bag containingmedia, collagenase and DNAse. Exemplary reagents are shown in thefollowing table:

TABLE 4 Disaggregation Media Animal/Human Available Raw Material DerivedSupplier Certificates Phosphate No Life CoA buffered saline TechnologiesLtd 2 mM Calcium No Sigma-Aldrich CoA Chloride DNAse 1 Approved RocheCoA (dornase alfa) medical product Products Ltd in the US CollagenaseBovine Nordmark CoA, CoO, type IV Arzneimittel TSE/BSE GmbH statement&Co KG BloodStor 55-5 No BioLife CoA (55% DMSO) Solutions

The bag is then heat sealed and its contents are disaggregated togenerate a homogeneous cell suspension containing tumor and TIL.Disaggregation is performed by a device, such as the Tiss-U-Stor devicedescribed herein, which runs a program to deliver a defined number ofrepeated physical compression events, with a defined compressionpressure over a defined duration to ensure enzyme access into the tumortissue thereby accelerating enzymatic digestion. The number of cycles,pressure, temperature, and duration are recorded for each individualtumor.

The homogenized cell suspension is then aseptically filtered using a 200pm filter (Baxter, RMC2159) and the filtrate passed aseptically into thecryopreservation bag. BloodStor 55-5 (Biolife Solutions, Bothell, WA) isaseptically added to achieve 5% DMSO. The cell suspension is thencryopreserved using the Tiss-U-Stor device with a defined coolingprogram, and the measured temperature profile is recorded for eachindividual cell suspension derived from each tumor portion. Thecryopreserved cell suspension is stored in vapor-phase of liquidnitrogen.

The cryopreserved cell suspension recommended storage condition is≤−130° C.

The cell suspension is transported from the clinical site to the GMPcell therapy manufacturing site by a qualified courier service packagedin a container validated to ensure the cryopreserved cell suspension ismaintained at ≤−130° C.

(Tiss-u-Stor)

Resected tumors are evaluated for weight and condition. For each tumorfragment, extraneous material is removed and the fragment weighed. ACS50N bag is opened, up to about 7 g of tumor is added and the bag isthen sealed. 15 ml of EDM digest medium is added to the bag with 2glgentamicin/amphotericin per ml EDM by syringe via needleless portfollowed by removal of air from the from the bag into the syringe.

The tumor tissue and disaggregation media in the disaggregation hag isplaced in the temperature controlled tissue disaggregator. Thetemperature is increased from ambient temperature to 35° C. at a rate of1.5° C./min and maintained at 35° C. for a total of about 45 minutesduring which time the disaggretor is active at 240 cycles per minute.

Once disaggregated the tumor material is filtered through an inlinefilter into a secondary freezing bag. 1.5 ml of Blood stor (DMSO) isinjected via a needleless port and air removed.

2 ml. of the suspention is withdrawn for testing.

For optional cryopreservation, the cryobag is loaded into a freezingcassette and the freezing cassette placed in the Via freeze. The Viafreeze is then cooled to −80° C., preferably directly from 35° C. to−80° C. at a rate of −2° C./min.

The frozen cryobag is then transferred to liquid nitrogen storage.

T1L Manufacture

Autologous tissue used for culturing in the United Kingdom (UK) shouldconform to HTA-GD-20, Guide to Quality and Safety Assurance for HumanTissue and Cells for Patient Treatment, established by the UK's HumanTissue Authority with suitable consent, Chain of Identity, Chain ofCustody and screening to confirm donors are negative for Hepatitis Bvirus, Hepatitis C virus, HIV-1 & 2, HTLV-1 & 2, and Syphilis.

Manufacturing involves outgrowth and expansion from a cryopreserved cellsuspension containing TILs and tumor cells derived from a resectedtumor. If the tumor is greater than about 7 g, the resection processgenerates multiple cryopreserved cell suspensions, where each cellsuspension derives from a 2-7 g tumor fragment. Typically, only one cellsuspension is needed to be thawed for 1 T1L outgrowth while theremaining cryopreserved cell suspensions remain in GMP control and heldat the recommended storage condition (vapor phase of liquid nitrogen).

In certain embodiments the cell suspension has been filtered afterdisaggregation, prior to cryopreservation. An exemplary manufacturingprocedure is shown in FIG. 66 . Exemplary Manufacturing Raw Materialsare provided in the following table:

TABLE 5 Raw Material Sourcing Raw Human/Animal Available MaterialDerived Supplier Certificates T Cell Medium Human and ThermoFisher CoA,CoO Animal Scientific Fetal Bovine Animal Life CoA, CoO Serum (FBS)Technologies Gentamicin/ No Life CoA Amphotericin Technologies B. 500×IL-2 (aldesleukin) Not Available Clinigen CoA Human AB Serum HumanValley CoA with Biomedical Origin MACS GMP CD3 No Miltenyi Biotec CoAOKT3 antibody Irradiated Buffy Coat Human SNBTS CoA Phosphate bufferedNo Life CoA saline Technologies Albumin (human) Human OctaPharma CoAwith 20% Origin CryoSure-DMSO No WAK-Chemie CoA, TSE Medical GmbH

T cell medium (TCM) contains Albumin (human), human Holo Transferrin,and animal origin cholesterol. The source plasma used to manufactureAlbumin and Transferrin are sourced from the USA and the donors aretested for adventitious agents.

Cholesterol is sourced from sheep woolgrease originating inAustralia/New Zealand, which complies with USDA regulations prohibitingruminant original material from countries with reported cases oftransmission spongiform encephalopathy (TSE).

Fetal Bovine Serum (FBS) is sourced from Australia/New Zealand incompliance with the USDA regulations prohibiting ruminant originalmaterial from countries with reported cases of transmission spongiformencephalopathy (TSE). The FBS is tested in compliance with 21 CFR part113.47, specifically including: bluetongue virus, bovine adenovirus,bovine parvovirus, bovine respiratory syncytial virus, bovine viraldiarrhea virus, rabies virus, reovirus, cytopathic agents, haemadsorbingagents. The FBS is heat inactivated at 56° C. for 30 minutes and triple0.1 μm filtered to provide two orthogonal viral removal steps.

Human AB Serum is sourced from Valley Biomedical, an FDA registeredestablishment (1121958). Each donor unit is tested for Hepatitis Bsurface Antigen (HBsAg), Hepatitis B Virus (HBV) Nucleic acidAmplification Test (NAT), anti-Human Immunodeficiency Virus (HIV) type 1and 2, HIV-1 NAT, anti-Hepatitis C Virus (HCV), HCV NAT, and a test forsyphilis by FDA approved methods. The serum is heat inactivated at 56°C. for 30 minutes and 0.1 μm filtered.

Irradiated Buffy Coat sourcing, preparation, shipment and storage: TheScottish National Blood Transfusion Service (SNBTS) screens donors,collects the blood component, prepares and irradiates buffy coats. TheSNBTS is licensed by the United Kingdom's Human Tissue Authority(license number 11018) in accordance with the Blood, Safety and QualityRegulations (2005) to procure, process, test, store and distributeblood, blood components and tissues.

Healthy donor screening meets or exceeds the requirements described inthe United States Code of Federal Regulations (CFR) Title 21 Part1271.75 with the exception that donors live in the United Kingdom. Whilethis presents a theoretical risk of sporadic Creutzfeldt-Jakob Disease(sCJD) or variant Creutzfeldt-Jakob Disease (vCJD), the United Kingdomhas a robust national surveillance program. The most recent annualreport, covering May 1990 to Dec. 31st 2018 (National CJD Research &Surveillance Unit, 2018), confirms the incidence of sCJD in the UK iscomparable to those observed elsewhere in the world, including countriesthat are free of bovine spongiform encephalopathy (BSE). There have beenno reported cases of vCJD in 2017 through Apr. 5th 2020, and only twocases identified nationally since Jan. 1st 2012 (NCJDRSU Monthly Report,2020). This rigorous surveillance network has eliminated transfusiontransmitted vCJD infections with none reported since 2007 (National CJDResearch & Surveillance Unit, 2018). Exemplary eligible donor testing(Table 7) meets 21 CFR Part 1271.85 requirements and adds Hepatitis Etesting which is not required.

TABLE 6 Exemplary donor screening (NHSBT) Pathogen SpecificationRequirement Hepatitis B, C & Not detected/Negative Every donation Evirus Human Not detected/Negative Every donation Immunodeficiency Virus(HIV) type 1 and 2 Syphilis Not detected/Negative Every donation Human TNot detected/Negative 1st donation and Lymphotrophic in selected Virus(HTLV) subsequent type 1 and 2 donations Malaria Not detected/NegativeTest performed T cruzi Not detected/Negative depending on the or IgGpositive donor's individual West Nile Virus Not detected/Negativecircumstances Cytomegalovirus Not detected/Negative (CMV) or IgGpositive

The licensed blood establishment prepares clinical grade irradiatedbuffy coats which are suitable to treat patients with severeneutropenia. To prepare the buffy coats, blood is centrifuged to formthree layers: the red blood cell layer, the buffy coat layer and theplasma layer. Buffy coats from 10 donors are irradiated with 25 to 50 Gyirradiation to arrest cell growth. The clinical grade irradiated buffycoats are prepared and shipped to the GMP manufacturing facility byovernight courier using a controlled temperature shipper including atemperature monitor. The shipment occurs one day before use in themanufacturing process.

Upon receipt, the buffy coats are held at 15-30° C. until use inmanufacturing.

Irradiated Feeder Cell Preparation

Buffy coats from up to ten unique donors are pooled, then centrifuged byFicoll gradient density centrifugation to harvest peripheral bloodmononuclear cells (PBMCs). Approximately 4×109 viable white blood cellsare resuspended in TCM supplemented with approximately 8% human ABserum, 3000 IU/mL IL-2 and 30 ng OKT-3 in a closed static cell culturebag. The PBMC are released per specification.

TABLE 7 Allogeneic PBMC stock specification Attribute Test methodAcceptance criteria Appearance Visual inspection ID label Identity Flowcytometry >85% viable CD45+ cells Viability Flow cytometry Reportresults Total viable Flow cytometry 2 to 4 ×10⁹ leukocyte content

The PBMC are also tested for sterility and mycoplasma. Immediately priorto starting step 3 (day 12, Fig. C), a sample of the formulated feedercell, including media, IL-2 and OKT3, is removed. This sample isincubated and analyzed on days 13, 17 and 18 to confirm that the feedercells do not expand.

Albumin (human), also known as Human Serum Albumin (HSA), is sourcedfrom US donors. All plasma donations are individually tested andnon-reactive to HBsAg, anti-HIV 1, anti-HIV 2, and anti-HCV antibodies.Each plasma pool is tested and found negative for HBsAg, anti-HIV 1,anti-HIV 2, and HCV-RNA by NAT. The HSA product is manufacturedaccording to GMP regulations fulfilling the production and testingcriteria of US and European Pharmacopoeia.

TIL Outgrowth

The cell suspension is seeded at approximately 0.25×10⁶ to 0.75×10⁶viable cells/mL into TCM supplemented with 10% FBS, 0.25 μg/mLAmphotericin B with 10 μg/mL Gentamicin (Life Technologies, GrandIsland, NY), and interleukin-2 (IL-2; aldesluekin) 3000 IU/mL (Clinigen,Nurnberg, Germany) and cultured in standard cell culture conditions (37°C., 5% CO2).

On day 5, half of the media is removed and replaced with TCMsupplemented with 10% FBS, 0.50 μg/mL Amphotericin B, 20 μg/mLGentamicin and 6000 IU/mL IL-2.

On day 7, if the cell concentration is >1.5×10⁶ viable cells/mL, the TILoutgrowth culture is diluted with three times the volume to maintainapproximately 0.1×106 to 2.0×106 viable cells/mL. If the cellconcentration is ≥1.5×106 viable cells/mL, half of the media isreplaced. In either option, the media is TCM supplemented with 10% FBS,0.50 μg/mL Amphotericin B, 20 μg/mL Gentamicin and 6000 IU/mL IL-2.

On day 10, if the cell concentration is >1.5×106 viable cells/mL, theTIL outgrowth culture is diluted with three times the volume to maintainapproximately 0.1×106 to 2.0×106 viable cells/mL. If the cellconcentration is <1.5×106 viable cells/mL, half of the media isreplaced. In either option, the media added is TCM supplemented with 10%FBS, 0.50 μg/mL Amphotericin B, 20 μg/mL Gentamicin and 6000 IU/mL IL-2.

TIL Activation

TILs are activated using an anti-CD3 antibody (OKT3) to provide a CD3specific stimulation when bound to the FC receptor of irradiated feedercells from allogeneic peripheral blood mononuclear cells (PBMCs). Thefeeders provide a natural source of additional co-stimulation to supportthe added anti-CD3 (OKT-3).

On day 12, 1 to 20×106 viable T cells from the TIL outgrowth Step 2 areadded to 2.0 to 4.0×109 viable irradiated feeder cells (Section 8.1.4.4)using approximately 30±10 ng/mL OKT3, approximately 8% Human AB Serumand 3000±1000 IU/mL IL-2. The TIL activation culture is incubated for 6days at standard cell culture conditions.

TIL Expansion

On day 18, the activated TILs continue expansion by aseptically addingthe activated TIL cell suspension into a bioreactor containing T cellmedia supplemented with approximately 8% Human AB Serum and 3000 IU/mLIL-2.

On day 19, the TIL expansion is provided a continuous feed of T cellmedia supplemented with 3000 IU/mL IL-2 until harvest.

TILs are harvested by washing the cells using SEFTA™. The cells areconcentrated by centrifugation then washed 2-4 times using phosphatebuffered saline (PBS) supplemented with 1% human serum albumin (HSA).The cells are then resuspended in PBS+1% HSA to approximately 50-60 mL.

The washed and concentrated cells are aseptically transferred into acryobag and a portion removed for lot release testing and retainedsamples. Cryoprotectant is added to achieve a formulated productof >5×109 viable cells suspended in approximately 10% DMSO and 8.5% HSAin PBS. A portion is removed for lot release testing and retainedsamples. The cryobag is cooled to −80° C.

TIL Manufacture Processes

The following table shows examples of process variations.

TABLE 8 Manufacturing Processes Process versions v1.0 v1.1 v1.2 ITIL-168Tumor disaggregation Manual Manual Tiss-U-Stor Tiss-U-StorDisaggregation Disaggregation Disaggregation Disaggregation StartingMaterial Fresh Cryopreserved Cryopreserved Cryopreserved TIL Outgrowth1-3 Weeks 1-3 Weeks 12 Days 12 Days Intermediate Hold CryopreservedCryopreserved Not Applicable Not Applicable Step TIL Recovery  3 Days  3Days Not Applicable Not Applicable Rapid Expansion 12 Days 12 Days 12Days 12 Days Phase Culture Extension 0-2 Days 0-2 Days Not ApplicableNot Applicable Final Product Fresh Fresh Cryopreserved Cryopreserved

The following table shows Drug Product Data

TABLE 8 Drug Product Data Product Lot Process Version Yield (× 10¹⁰)Viability Percent CD3⁺ Cells TIL001 1.0 1.1 82 N/A TIL003 1.0 2.2 94 98TIL005 1.0 2.0 96 N/A TIL012 1.0 3.2 95 98 TIL013 1.0 2.1 80 92 TIL0141.0 4.4 91 95 TIL015 1.0 6.4 91 97 TIL016 1.0 5.5 93 96 TIL027 1.0 3.895 97 TIL032 1.0 3.7 92 99 TIL035 1.0 6.4 96 90 TIL037 1.0 2.6 92 97TIL038 1.0 1.3 83 98 TIL039 1.1 1.2 80 93 TIL040 1.0 5.3 93 97 TIL0411.0 3.2 93 98 TIL043 1.0 4.8 93 98 TIL054 1.1  0.82 86 91 TIL065 1.1 3.494 97 TIL067 1.2 3.0 91 97 TIL073 1.0 5.4 92 98 TIL077 1.2 1.0 91 97TIL078 1.2 3.4 99 98 E2 1.2 3.5 86 97 E3 1.2 1.8 80 96 E4 1.2 1.0 88 93E5 1.2 4.1 98 100 

Comparing cryopreserved and fresh cell suspensions, representativeyields were consistent as demonstrated by similar drug substance yield(FIG. 67A), viability (FIG. 67B), and percent T cells (FIG. 67C).

Optimization of Cryopreservation—As a surrogate to tumor material,isolated PBMCs were digested using the Tiss-U-Stor process andmaterials. Commercial cryopreservation agents (CPAs) were evaluatedacross a range of conditions to determine which reagent maximizedpost-thaw viability (FIG. 68 ). The post-thaw viabilities of two CPAs,Cryostor10 and Stem Cell Banker DMSO free, were similar. CryoStor basedDMSO was then compared with Bloodstor 55-5, a DMSO basedcryopreservative, and the higher concentration BloodStor product wasselected since it was more concentrated thus allowing for a smallercryobag. Cryopreservation was then compared following a protocol thateither held the material at 4° C. for 10 minutes, then decreased thetemperature at a rate of −1° C./min or decreased from 35° C. to −80° C.directly at a rate of −2° C./min. Post-thaw viability was similarbetween the two cryopreservation protocols used (FIG. 69 ).

During cooling, ice nucleation releases heat. Undercooling, a phenomenonwhere the released heat appears to warm the solution, is associated withlower post-thaw recoveries. Temperature data was recorded from testarticles during cryopreservation using both protocols (FIG. 70 ).Undercooling was observed in both independent runs using the −1° C./minprotocol, whereas the −2° C./min cooling protocol recorded noundercooling event once, and in the second independent run, anundercooling event was observed to release less heat relative to thealternative protocol (FIG. 70 ).

The cryopreserved DP is transferred to vapor phase LN2 for storage andtransport at ≤−130° C.

Sample sterility is tested and retained samples are frozen using aCoolcell® (Biocision, Larkspur, CA) at −80° C. then transferred to vaporphase LN2 for storage purposes.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1

FIG. 39 shows an embodiment of bag 400 during use. As depicted, bag 400is secured by a securing element such as clamp 402 within device 404such as tray 406. Tissue 408 is visible through a transparent side ofbag 400. Tubing 410 is coupled to bag 400.

Example 2

FIG. 40 depicts an embodiment of bag 420 for use in the invention asdescribed herein. As depicted, bag 420 is secured by a securing element422 from device and tray 424. Tissue material 424 is visible throughtransparent side of bag 420. Tubing 426 is coupled to bag 420. As showna position of bag 400 within tray 406 is further secured using fixationelement 428, in particular tape. Tissue 424 is visible throughtransparent side of hag 420. As shown in FIG. 40 , bag may include ports430 to access the interior of bag and/or tissue 424.

Example 3—Disaggregation and Cryopreservation

TIL075 was manufactured from metastatic melanoma tumor pieces (samples).The tumor samples were weighed and processed as follows. Si=1.4 g. Siwas disaggregated by an automated procedure. S2=19.4 g. S2 was divided,one portion (about 7.7 g) was disaggregated by an automated procedureand the second portion (about 12 g) was disaggregated manually.

Manual disaggregatrion: The tumor sample was cut into smaller 2-4 mm³pieces and added to a bottle containing 80 ml of digestion media withantibiotics. The bottle was placed on a shaker and disaggregatedovernight (about 14 hours) at 37° C. The digest was then filteredthrough netwells and 100 μM cell strainers into Falcon 50 tubes. 10% ofthe filtered digest was set aside for sterility testing. The remainderwas centrifuged and resuspended in 12 ml of CS10 and divided into 12cryovials.

Tiss-U-Stor disaggregation: Two CS50N bags were opened with sterilescissors, cutting the end without ports. The Si 1.4 gm sample and the7.7 gm portion of S2 were placed in the CS50N bags and the bags sealed.15 ml of disaggregation media and 30 ul of antibiotics are combined andadded to each of the sealed bag using a syringe through needleless portsof the bags. The bags were transferred to a Tissue Disaggregator loadedin a ViaFreeze and the disaggregation protocol was initiated. TheDisaggregation protocol called for a temperature increase from ambientat a rate of 1.5° C./min to 35° C., and a temperature hold at 35° C.while the disaggregator was active. The disaggregator speed was set to240 cycles/min. The temperature of the ViaFreeze remained at 35° C.thereafter until the cryopreservation step.

The bag setup includes a direct connection by tubing through an inlinefilter to a secondary cryobag. The disaggregated material in the CS50hag was filtered into the cryobag and the tubing connection sealed. 1.5ml Blood-stor (DMSO) was slowly added through a needleless port of thecryobag, the bag was placed in a cassette designed for optimal heattransfer, and the cassette was placed back in the ViaFreeze in place ofthe disaggregator.

A post-disaggregation cryopreservation protocol was engaged. The freezecycle ramped the temperature of the ViaFreeze from 35° C. at −2° C./minto −80° C. Frozen bags were transferred to liquid nitrogen storage.

Example 4—Disaggregation and Cryopreservation

TIL077 was manufactured from metastatic melanoma tumor pieces (samples).The tumor samples were weighed and processed as follows. Si=4.6 g.S2=4.6 g.

Tiss-U-Stor disaggregation: Two CS50N bags were opened with sterilescissors, cutting the end without ports. The Si=4.6 gm sample and theS2=4.6 gm sample were placed in the CS50N bags and the bags sealed. 15ml of disaggregation media and 30 ul of antibiotics are combined andadded to each of the sealed bag using a syringe through needleless portsof the bags. The bags were transferred to a Tissue Disaggregator loadedin a ViaFreeze and the disaggregation protocol was initiated. TheDisaggregation protocol called for a temperature increase from ambientat a rate of 1.5° C./min to 35° C., and a temperature hold at 35° C.while the disaggregator was active.

The disaggregator speed was set to 240 cycles/min. The temperature ofthe ViaFreeze remained at 35° C. thereafter until the cryopreservationstep. FIG. 71 shows disaggregation records.

The bag setup includes a direct connection by tubing through an inlinefilter to a secondary cryobag. The disaggregated material in the CS50bag was filtered into the cryobag and the tubing connection sealed. 1.5ml Blood-stor (DMSO) was slowly added through a needleless port of thecryobag, the bag was placed in a cassette designed for optimal heattransfer, and the cassette was placed back in the ViaFreeze in place ofthe disaggregator.

A post-disaggregation cryopreservation protocol was engaged. The freezecycle ramped the temperature of the ViaFreeze from 35° C. at −2° C./minto −80° C. FIG. 71 shows cryopreservation records. Frozen bags weretransferred to liquid nitrogen storage.

Example 5—Disaggregation and Cryopreservation

T1L078 was manufactured from metastatic melanoma tumor pieces (samples).The tumor samples were weighed and processed as follows. Si=11 g. S2=2g.

Tiss-U-Stor disaggregation: Two CS50N bags were opened with sterilescissors, cutting the end without ports. The tumor material was dividedand 6.4 gm of sample was placed in each of two CS50N bags and the bagssealed. 15 ml of disaggregation media and 30 μl of antibiotics arecombined and added to each of the sealed bag using a syringe throughneedleless ports of the bags. The bags were transferred to a TissueDisaggregator loaded in a ViaFreeze and the disaggregation protocol wasinitiated. The Disaggregation protocol called for a temperature increasefrom ambient at a rate of 1.5° C./min to 35° C., and a temperature holdat 35° C. while the disaggregator was active. The disaggregator speedwas set to 240 cycles/min. The temperature of the ViaFreeze remained at35° C. thereafter until the cryopreservation step. FIG. 72 showscryopreservation records

The bag setup includes a direct connection by tubing through an inlinefilter to a secondary cryobag. The disaggregated material in the CS50bag was filtered into the cryobag and the tubing connection sealed. 1.5ml Blood-stor (DMSO) was slowly added through a needleless port of thecryobag, the bag was placed in a cassette designed for optimal heattransfer, and the cassette was placed back in the ViaFreeze in place ofthe disaggregator.

A post-disaggregation cryopreservation protocol was engaged. The freezecycle ramped the temperature of the ViaFreeze from 35° C. at −2° C./minto −80° C. FIG. 72 shows cryopreservation records. Frozen bags weretransferred to liquid nitrogen storage.

Example 6—Disaggregation and Cryopreservation

TILOS I was manufactured from metastatic melanoma tumor pieces(samples). The software was updated to include disaggregation andcryopreservation in a single protocol. FIG. 73 shows disaggregation andcryopreservation records. As in the prior examples, the disaggregatorwas active for about 53 min. (FIGS. 73A, 73B). The disaggregated tissuewas transferred from the disaggregation bag through a filter to thecryobag and returned to the ViaFreeze for cryopreservation within about90 min. from the start of the disaggregation process at which timecryogenic cooling was initiated.

Example 7—Manufacture from Vials

TABLE 9 Cell cryopreservation and thawing Reagents/Materials ReagentManufacturer Catalog # Media depending on cell type NA NA DPBS SigmaD8537-500ML 15 mL Centrifuge Tube VWR 339650 Stripette 10 mL CorningCLS4101  Stipette 25 mL Corning CLS4251  Stripette 5 mL Corning CLS4051Tips 1000 μL filtered StarLabs S1182-1730 Trypan Blue Sigma T8154-100ML

TABLE 10 Equipment Serial #/ Description Manufacturer Part # Asset#Powerpette pro 1-100 mL VWR 452-8344 NA Pipette ErgoOne Star LabsS7110-1000 NA 100-1000 μL Megafuge 40R Centrifuge Hereus 7500451841536283 Hemacytometer Hawksley HC002 NA Water Bath 12L VWR 462-0557BP1912001 IncuSafe CO₂ Incubator PHCBI MC O-170AIC-PE NA

Cryovials were removed from liquid nitrogen and placed in a 37° C. waterbath until the cell suspension is just melted. Cell suspensions wereplaced in a 15 mL falcon and topped up with PBS up to 10 mL, andcentrifuged at 400 g for 10 minutes. The supernatant was decanted.

For cell culture, cell pellets were resuspended in pre warmed media,initially in a small volume i.e. 2 to 3 mL. Adherent cell lines (i.e.tumor lines, HEK 293s) were added to tissue flasks with media inaccordance with the following table. Non adherent cell lines (i.e. Tcells, TILs, Jurkat cells) were plated at a density of 0.5 to 1×10⁶cells per mL. Flasks were placed in a humidified 37° C. incubator andmedia replaced every 2-3 days.

TABLE 11 Cell seeding densities for adherent cells in different vesselsVessel/flask type Seeding density Media volume mL 24 well 0.1 × 10⁶ 0.5to 1    6 well 0.5 × 10⁶ 2 to 4 T25 0.7 × 10⁶ 4 to 6 T75 2.1 × 10⁶ 12 to15 T 150 4.4 × 10⁶ 25 to 30

Example 8—Manufacture from Cryopreserved Disaggregated Tumors

Manufacturing Process

Thawing Starting Materials

The VIAThaw CB1000 Thawing system was used to control heating ofcryopreserved samples stored in cryo-bags. Cryopreserved cell suspensionwas thawed, then diluted in T-cell media (TCM) manufactured by LifeTechnologies (Paisley, United Kingdom). TCM contains 80% Rosewll ParkMemorial Institute (RPMI) 1640 medium and 20% AIM V. The cell suspensionwas filtered through a 70- to 100-μm filter and centrifuged, and thesupernatant removed. The cell pellet was resuspended in TCM supplementedwith 10% irradiated Fetal bovine serum (FBS) (Life Technologies,Auckland, New Zealand).

A disaggregated, cryopreserved tumor (about 16.5 ml) in an Origin CS50bag was placed in the thawing tray of a VIAThaw CB1000 Thawing Systemand warmed to about 0° C.

Example 9—Potency

A co-culture-based potency method quantitates the percentage of T cellsactivated by an OKT3-expressing target cell line. The TIL productmechanism of action in vivo involves TIL peptide presentation throughpMEIC-HLA, which binds to the TCR in vivo. The potency assay quantifiesthe percentage of potent T cells, defined as viable T cells positive foreither CD137, IFN-γ, TNFα, or CD107a divided by the total viable T cellswhen specifically activated by co-culture with a K562 cell lineexpressing the OCT3 antigen-binding domain. Markers used to quantitate Tcell potency include DRAQ7, CD45, CD2, CD107a, CD137, TNF-α, and IFN-γ.

To measure the potency, ITIL-168 DS cells are co-cultured forapproximately 5 hours using 1 of 3 cell lines: Condition 1—Nostimulation—background cell activity; Condition 2—K562 cellline—background TCR-independent reactivity; Condition 3—K562 cell lineexpressing an ScFv against OKT-3—TCR-induced T-cell stimulation.

The cultured cells are analysed by flow cytometry and gated on viablewhite blood cells to quantitate the T cells that express at least 1 of 4activation markers. For stability tests, cryopreserved DP cells arethawed, washed, and rested overnight.

ITIL-168 TCR potency is calculated as follows: Step 1) the % potency dueto non-specific stimulation is obtained from Condition 2; Step 2) the %potency due to CD3 specific and non-specific stimulation is obtainedfrom Condition 3; Step 3) the % potency due to CD3 specific stimulationis calculated as Condition 3-Condition 2.

For both Condition 2 and Condition 3, the % potent result is 100% minusthe percentage of all T cells that are CD137-/IFN-γ-/TNFα-/CD107a- (i.e.background). This population does not produce at least one marker.

Example 10—TIL Outgrowth and Rapid Expansion

The TIL manufacturing process begins after the tumour resection,disaggregation, cryopreservation, and optional packaging and shipment.shipment packaging, and shipment from the Tumour Processing Huh toInstil's manufacturing facility in a qualified shipper under controlledconditions. Th cryopreserved tumor and T cells are thawed usingcontrolled conditions, and diluted in T cell media (TCM) composed of 80%Roswell Park Memorial Institute (RPMI) 1640 medium and 20% AIM V,supplemented with 10% FBS, Amphotericin B, Gentamicin, Vancomycin, andIL-2 (herein referred to as ICMT).

The cells are washed by centrifugation in closed bags, resuspended inICMT and samples are taken for cell counts. Cell suspension is seededinto culture bags with ICMT targeting 0.25×10⁶ viable cells/mL andincubated under controlled conditions up to Day 8 of the process. On Day8, samples for cell counts are taken and an equal volume of ICMT isadded to the culture bag and incubated under controlled conditions. OnDay 11, cell counts are taken and an equal volume of ICMT is added tothe culture bag and incubated under controlled conditions. On Day 13,cell counts are taken, and TILs are concentrated by centrifugation in abag to provide between 1×10⁶ to 20×10⁶ viable T cells.

Also on Day 13, the 1×10⁶ to 20×10⁶ viable outgrown TILs are activatedusing anti-CD3 and irradiated feeder cells (allogenic PBMCs) with TCMcontaining 8% Human AB serum and IL-2 (herein referred to as WTCM). TheTIL activation culture is incubated for up to 6 days under controlledconditions in static culture bags. On Day 19 of incubation, cell countsare performed and activated TILs are seeded into a bioreactor containingWTCM. Cells are incubated for up to 6 days under controlled conditions.On Day 20, TIL expansion is provided a continuous feed of TCMsupplemented with IL-2 until harvest target dose is achieved before orby Day 27 of the process.

Once harvest dose is achieved, the cells are counted, washed andconcentrated by centrifugation in phosphate buffered saline (PBS)supplemented with 1% human serum albumin (HSA). The TILs in the drugproduct (DP) bag are then cooled to 2-8° C. and formulated 1:1 withcryoprotectant containing 16% HSA and 20% DMSO to provide a finalformulation of DP in PBS containing 8.5% HSA and 10% DMSO. Samplevolumes are removed for lot release testing, reference and back-upsamples.

Formulated DP is cryopreserved in a CRF using a pre-defined programuntil the product reaches a specified temperature. The cryopreserved DPis then transferred to liquid nitrogen storage before transportation at≤−130° C. to clinics for administration.

TABLE 12 Equipment Equipment/Supply Manufacturer Model or Catalog #Leukosep ficoll tubes Greiner Bio-One Lrd 227288 PermaLife Cell CultureBag, Origen Biomeical Inc PL325-2G 325 ml Cell culture expansion hagCharter Medical Ltd. EXP-1L WAVE 10L bag Cytiva 29-108-1-7173 CT800.1Sefia kit Cytiva 2000 I

TABLE 13 Reagents Reagent Manufacturer Catalog # Lot # Expiry # T-cellmedia Life 04196658P 2021537 31.Aug.2020 Technologies Gamma-irradiatedFBS Life 01190005H- 2225231RP 31.May.2024 Technologies RESERVE 2-2YBT2DSProleukin manufacturer Clinigen Proleukin 801313T 31.Dec.2020 vial(IL-2) Group PLC Aliquoted 11-2 stock N/A N/A CTU- 31.Aug.2020IL2/02/09/2019 Gentamicin/Amphotericin Life RO1510 2217613 30.Mar.2021solution (500x) Technologies Vancomycin Bowmed N/A 90260 28.Feb.2021manufacturers vial Ibisqus Vancomycin aliquot N/A N/A CTU-12-06-202028.Feb.2021 (50 mg/ml) Gamma-irradiated human Gemini Bio- 100-812GH12Y00K 30.Sept.2020 AB serum Products LLC OKT-3 manufacturers Miltenyi170-076- 6200108211 17.Oct.2020 vial (1 g/ml) Biotec Ltd 116 AliquotedOKT-3 N/A N/A CYU- 17.Oct.2020 OKT3/05/05/2020 20% Human serum Nova68982- M848B6661 27.Nov.2021 albumin Biologics Inc 0633-02 CryoSure DMSOWAK-Chemie WAK- USP8C1S 28.Feb.2022 Medical DMSO-50 GmbH

Example 11

Full-scale runs were performed under GMP conditions. The ITIL-168process used in these runs included the use of cryopreserved tumordigest, a target of 0.25×10⁶ viable cells/mL seeding for the TILoutgrowth stage (stage 1), continuous processing from the TIL outgrowthto TIL rapid expansion phase (REP), and automated formulation of thefinal product and cryopreservation of the final drug product.

ITIL-168 is a tumor-infiltrating lymphocyte (TIL) therapy for thetreatment of adult patients with advanced melanoma who have relapsedfrom or are refractory to at least one prior line of therapy. ITIL-168consists of a single infusion of autologous T cells isolated andexpanded ex-vivo from a patient's cancer tissue and administeredintravenously. Process improvements have been identified and implementedover time, the improved process referred to as ITIL-168. Tablesummarizes process variations. me and implements been identified andimplemented over time, the improved process referred to as ITIL-168.Table summarizes process variations. Me and implements.

TABLE 14 Summary of Manufacturing Process Developments Unit ProcessOperation/ Step Change MS v.10 MS v1.1 UTIL-01 ITIL-168 Process TumourTumour Manual Manual Automated Automated Digest Disaggregationdisaggregation disaggregation disaggregation disaggregation Preparationin bottles in bottles in bags fusing in bags fusing the Tiss-n-for theTiss-n-for device) device) Tumour Digest Non- Non- CryopreservedCryopreserved Formulation cryopreserved cryopreserved TIL Culture Openprocess Open process Open process Closed process in bags OutgrowthVessels for in plates in plates in plates Tumour Digest Seeding Targetof 1 × Target of 1 × Target of 0.5 × Target of 0.25 ×  

Density

 viable

 viable

 viable viable cells/mL cells/mL cells/mL cells/mL Cell Count

Flow Flow cytometry Flow cytometry Test Method cytometry MateriałGentamycin & Gentamycin & Gentamycin & Gentamycin, Amphotericin BAmphotericin B Amphotericin B Amphotericin B, & Vancomycin MaterialHeatin activated Heat inactivated Heat inactivated Heat inactivated andand 0.1 μm and 0.1 μm and 0.1 μm 0.1 μm filtered filtered FBS filteredFBS filtered FBS Irradiated FBS TIL REP Material Heat inactivated Heatinactivated Heat inactivated Heat inactivated and and 0.1 μm and 0.1 μmand 0.1 μm 0.1 μm filtered Human AB filtered Human filtered HumanIrradiated Human AB donors AB donors AB donors donors TIL Post TIL Holdstep with Hold step with Continuous Continous processing OutgrowthOatgrowth, Cryopreser- Cryopreser- processing without to REP Cryopreser-vation and vation and without cryopreservation vation. Thaw/ 1-3 dayspost 1-3 days post cryopreser- wash and thaw recovery thaw recoveryvation Recovery Harvest to Drug Product Haemonetics HaemoneticsHaemonetics Cytiva Sefia S-2000 Drug Cell Saver 5 Cell Saver 5 CellSaver 5 (Automated Product (Manual (Manual (Manual formulation toFormulation formulation to formulation to formulation to 110 mL) 270 mL)270 mL) 270 mL) Drug Drug Product Non- Non- Cryopreserved CryopreservedProduct cryopreserved cryopreserved Formulation

indicates data missing or illegible when filed

An overview of the ITIL-168 manufacturing process used in the twoprocess development runs is shown in Table 15. The two processdevelopment runs, labelled as Run 1 (TIL065) and Run 2 (Biopartners9251), were performed at full scale under GMP conditions and used excesstumor gathered from a patient and tumor sourced from thevendor—Biopartners, respectively.

During these two process development runs, in-process testing forbioburden and final product sterility, endotoxin, mycoplasma andappearance tests were not performed, as these runs were primarilyintended to evaluate manufacturing process performance and productquality following the process improvements, as well as serve as trainingruns for the manufacturing operators, under GMP conditions prior to theprocess verification runs.

TIL outgrowth and REP were performed as in Example 10 using thematerials shown in Table 12 and Table 13.

For both runs (Run 1 and Run 2), total CD3+ cell counts were measured ondays 1, 8, 11 and 13 for the TIL outgrowth stage or stage 1, and on days13, 19, 22 and 25 for the TIL Rapid Expansion Phase (REP) or stage 2,per the batch manufacturing record (BMR). FIGS. 76A and 76B show thetotal CD3+ cell count for the two runs throughout the TIL outgrowthstage (stage 1) and TIL REP stage (stage 2), respectively. Data shown inFIG. 76B demonstrates that for both runs, >1×10¹⁰ CD3+ cells wereachieved by the end of the REP stage resulting in both lots meeting thedose acceptance criteria of 5×10⁹ to 5×10¹⁰ CD3+ cells.

Viability (percentage of viable CD3+ cells) was also measured for bothruns on days 1, 8, 11, 13 and 25. FIG. 76C shows that the viabilityincreased during the manufacturing process and towards the end of REPstage and both runs met the final product criteria of >70%.

Fold expansion for the rapid expansion phase (REP) was calculated fromthe cell count data, for the two runs. Additionally, final productquality attributes such as dose, viability, potency, T cell phenotypeand T cell subsets were also evaluated for the two process developmentruns.

Data presented in Table 16 demonstrates that following the processimprovements, the ITIL-168 manufacturing process performs similarly tothe historical process and results in final product quality attributesthat meet the specification requirements.

TABLE 16 ITIL-168 manufacturing process performance and product qualityattributes Fold Expansion Dose during REP (Total viable ViabilityPotency¹ Run (Absolute) CD3+ cells) (%) (%) Acceptance Criteria/ NA 5 ×10⁹ to ≥70 ≥40 Specification 5 × 10¹⁰ Requirements Historical 395 − 75267.90 × 10⁹ to 80 − 99 Historical Range Observed (n = 22) 6.25 × 10¹⁰ (n= 23) retains in the (n = 23) process of being tested Run 1 1350 3 ×10¹⁰ 90 63.2 Run 2 1700 2 × 10¹⁰ 88 65.2 ¹Potency is calculated as thefrequency of all viable CD2+ cells that are positive for one or more ofCD137, CD107a, TNF-α and IFN-γ

Example 12—Administration

Therapy

Subjects received a lymphodepleting chemotherapy regimen ofcyclophosphamide and fludarabine. The therapy is designed to reduce theinfluence of suppressive cells such as regulatory T cells and toincrease the expression of lymphocyte growth-promoting cytokines (e.g.,IL-7 and IL-15). A hydration regimen was initiated prior to and duringlymphodepleting chemotherapy. Antimicrobial and antifungal prophylaxiswas initiated prior to starting lymphodepleting chemotherapy. Fever andneutropenia were assessed and managed. Non-steroidal anti-emetic therapywas commenced prior to lymphodepleting chemotherapy and continued asnecessary.

Lymphodepleting chemotherapy was administered as follows. The doses ofcyclophosphamide and fludarabine administered was calculated basedassessment of body weight taken at baseline visit. In obese subjects(body mass index>35), the practical body weight was used. The dose ofcyclophosphamide is based on weight, and the dose of fludarabine isbased on body surface area. Doses may be rounded up or down inaccordance with practices on dose banding. The following table showsrecommended doses, routes of administration, infusion volumes, andduration:

TABLE 17 Lymphodepleting Chemotherapy Regimen Day Drug Dose RouteAdministration −7 Fludarabine 25 mg/m² IV In 10-100 ml 0.9% NaCl overapprox. 30 mins. Cyclophosphamide 60 mg/kg IV In 500 ml 0.9% NaCl overapprox. 1 hr. −6 Fludarabine 25 mg/m² IV In 10-100 ml 0.9% NaCl overapprox. 30 mins Cyclophosphamide 60 mg/kg IV In 500 ml 0.9% NaCl overapprox. 1 hr. −5 Fludarabine 25 mg/m² IV In 10-100 ml 0.9% NaCl overapprox. 30 mins −4 Fludarabine 25 mg/m² IV In 10-100 ml 0.9% NaCl overapprox. 30 mins −3 Fludarabine 25 mg/m² IV In 10-100 ml 0.99% NaCl overapprox. 30 mins −2 Restday −1 Restday

TABLE 18 Fludarabine Dose Adjustment Creatinine clearance (measured byCockeroft-Gault formula) Fludarabine dose >/= 70 mL/min  25 mg/m² 51-69mL/min 20 mg/m²

Subjects were premedicated with antihistamine and acetaminophen prior toTIL infusion. The contents of an infusion bag were infused using anon-leukodepleting filter (e.g. in-line/tubing filter of >/=170microns). Subjects received up to 8 doses of intravenous IL-2 forpost-infusion support. IL-2 was administered after the completion of TILinfusion beginning on day 0 and continuing through day 4.

Example 13—Treatment Results

A total of 44 patients with metastatic cutaneous melanoma underwenttumour resection and initiation of TIL Outgrowth manufacturing (stage1). Of these 44 patients, 42 individual patient lots completed stage 1,with 2 failed attempts. Thirty-one patient lots were taken forward toREP manufacturing (stage 2). One lot failed the TIL outgrowth stage 1manufacturing and a revised stage 1 manufacturing process wasimplemented which enabled successful stage 2 manufacturing. The patientwas subsequently treated. The remaining 12 lots were not selected forinitiation of REP for the following reasons: 8 were due to intercurrentclinical deterioration of patient status rendering them unfit for TILtherapy, 2 patients no longer required TIL due to clinical improvementon other therapies, 1 patient was unable to secure funding for thetreatment, and 1 lot failed manufacturing due to lack of tumour tissueon the excised specimen. Four patient lots were manufacturedsuccessfully, however, the patients were deemed clinically unfit for theTIL therapy and hence were not treated.

Of the 44 tumours that were resected, 2 failed manufacturing, yielding a95% manufacturing success rate. Twenty-seven patients were treated withTIL products made utilizing the standard manufacturing process. At thetime of completion of TIL manufacturing, 6 of these patients were deemedclinically unfit for the full treatment regimen and received markedlylower doses of conditioning chemotherapy and post-infusion IL-2 and weretherefore excluded from the analysis. One patient had a tumour resectionwhich did not meet the criteria to initiate the standard TIL outgrowthmanufacturing step (stage 1). Therefore, a modified stage 1 wasinitiated which did enable a rapid expansion protocol (stage 2) andfinal product formulation, albeit at a very low final cell dose(1.7×10⁹). Because this product was produced using a modifiedmanufacturing process and yielded a low dose of cells, it was notconsidered representative of the MS license process and therefore theclinical data was excluded from the analysis.

The demographics, baseline patient characteristics, treatment detailsand disposition, and clinical efficacy and safety outcomes of theremaining 21 patients were collected and analysed. By the analysiscutoff date, these patients had a median potential follow-up time of52.2 months (range: 4.6, 98.8 months) from the TIL infusion date.

Among these 21 patients, the majority (71%) were male, and the medianage at the time of TIL treatment was 45 years (range: 16, 68). Atbaseline, all patients had stage IV metastatic cutaneous melanoma with amedian of 39 months since original diagnosis of melanoma (range: 8,177). A majority (67%) of patients had lesions reported in more than 3disease sites, including 7 (33%) with brain metastasis documented at thetime of the TIL treatment. The median number of prior systemic therapieswas 2 (range: 1, 9). Fifty-two percent (52%) of the patients had a BRAFmutation, all of whom had received and progressed on a BRAF inhibitorwith or without a MEK inhibitor. All but two patients (90%) had at leastone prior checkpoint inhibitor with 12 (57%) having received a PD-1inhibitor (either nivolumab or pembrolizumab). Additionally, 8 (38%)received ipilimumab and either nivolumab or pembrolizumab given insequence and 4 (19%) received ipilimumab and nivolumab concurrently.Prior to the tumour resection for T1L production, 20 (95%) had relapsedor refractory progressive melanoma, and 1 (5%) ceased treatment prior toTIL therapy due to intolerability.

Immediately prior to receiving TIL, 10 (48%) of the patients hadelevated serum lactose dehydrogenase (LDH) levels with 7 (33%) between 1and 2 times of the upper limit of the normal range (ULN) and 3 (14%)higher than 2 times of ULN. Baseline tumour burden as measured in thesum of lesion dimensions (SLD) of the target lesions was available for20 patients; the median baseline SLD was 100 mm (range: 13, 281).

TIL Treatment

All 21 patients received 2 doses of cyclophosphamide and 5 doses offludarabine as conditioning chemotherapy prior to the TIL infusion. Themedian total number of TIL cells infused was 31.9×10⁹ (range: 7.9×10⁹,62.5×10⁹). The median total number of IL-2 doses was 8 (range: 4, 11).Patients remained in the hospital for a median of 10 days (range: 7,15). Three (14%) patients were admitted for ICU during the treatmentperiod.

Clinically significant AEs during the TIL treatment period werereported. Common AEs (≥10%) reported during the conditioningchemotherapy period included neutropenia (43%) and nausea (19%) and arebroadly consistent with the side effect profile of these chemotherapyagents.

Common AEs with onset post TIL infusion included thrombocytopenia (62%),pyrexia (57%), rigors (43%), tachycardia (29%), neutropenia (29%),pulmonary oedema (24%), vascular leak (24%), rash (19%), atrialfibrillation (14%), cardiovascular instability (14%), chest infection(14%), and oedema (14%) (Table 19). These AEs are consistent with thosereported in other TIL trials (Dafni et al, 2019; Rohaan et al, 2018).

The patient whose manufacturing process failed stage 1 but was treatedwith a product generated from a modified manufacturing process died onday 6 following TIL therapy due to extensive tumour burden exacerbatedby renal failure, fluid overload and possible sepsis.

TABLE 19 AEs With Onset Post TIL Infusion ( All Treated Subjects) AllTreated Subjects AE Term-n (%) (N = 21) Thrombocytopenia 13 (61.9)Pyrexia 12 (57.1) Rigors  9 (42.9) Neutropenia  6 (28.6) Tachycardia  6(28.6) Pulmonary oedema  5 (23.8) Vascular leak  5 (23.8) Rash  4 (19.0)Atrial Fibrillation  3 (14.3) Cardiovascular instability  3 (14.3) Chestinfection  3 (14.3) Oedema  3 (14.3) Confusion 2 (9.5) Hypokalaemia 2(9.5) Hypotension 2 (9.5) Neurological deficit 2 (9.5) Renal impairment2 (9.5) Respiratory sepsis 2 (9.5) Seizure 2 (9.5) Sepsis 2 (9.5)Vitiligo 2 (9.5) Weight gain 2 (9.5) Wheezing 2 (9.5) Cough 1 (4.8)Diarrhoea 1 (4.8) Dysphasis 1 (4.8) Engraftment syndrome 1 (4.8)Hallucinations 1 (4.8) Lethargy 1 (4.8) PICC line infection 1 (4.0)Pleural effusion 1 (4.8) Pneumonia 1 (4.8) Pneumonitis 1 (4.8)Respiratory problems 1 (4.8) Tachypnoea 1 (4.8)

Peripheral blood counts were measured during the treatment period. Atrend of decrease in neutrophils, platelets, lymphocytes, white cellcount, and haemoglobin was observed at the time of initiation ofconditioning chemotherapy. Blood cell counts and haemoglobin levelsgenerally reached their nadirs 1-4 days after the TIL infusion. Theblood count recovery to baseline levels was generally observedapproximately 7 days after the TIL infusion date.

A recent change in the manufacturing process was implemented to improverobustness and enable multicentre clinical trials with centralizedmanufacturing. In this update, digested tumour material is cryopreservedto prolong stability. Importantly, in the four patients treated withproducts made with up-front cryopreservation, the AE profile observedwas broadly consistent with the other patients treated in the series(Table 20) and with that reported in clinical trials of other TILproducts.

TABLE 20 AEs With Onset Post TIL Infusion (Subjects Treated with Cryo-InProducts) All Treated Subjects AE Term-n (%) (N = 4) Thrombocytopenia 4(100)  Pyrexia 2 (50.0) Rash 2 (50.0) Rigors 1 (50.0) Hypotension 1(25.0) Renal impairment 1 (25.0) Vascular leak 1 (25.0) Vitiligo 1(25.0)

Fifteen of the 21 patients underwent disease assessments by serial CTand/or MRI scans that included radiological measurements of targetlesions. Among these patients, the quantitative response rate(confirmation of response not required) was 53%, including 2 (13%)patients who achieved a CR and 6 (40%) who achieved a PR (Table 21).

TABLE 21 Summary of Best Overall Response (Efficacy Evaluable AnalysisSet) Efficacy Evaluable Analysis Set (N = 15) Best Overall ResponseComplete Response (CR) 2 (13.3) 95% Cl (Clopper-Pearson method) 1.7,40.5 Partial Response (PR) 6 (40.0) 95% Cl (Clopper-Pearson method)16.3, 67.7 Stable Disease (SD) 3 (20.0) 95% Cl (Clopper-Pearson method)4.3, 48.1 Progressive Disease (PD) 4 (26.7) 95% Cl (Clopper-Pearsonmethod) 7.8, 55.1 Response Rate (CR + PR) 8 (53.3) 95% Cl(Clopper-Pearson method) 26.6, 78.7 Disease Control Rate (CR + PR + SD)11 (73.3)  95% Cl (Clopper-Pearson method) 44.9, 92.2

The response rate inclusive of all patients based on both quantitativeand qualitative response was 57%, including 3 (14%) who achieved a CRand 9 (43%) who achieved a PR. Two additional patients had developedresistance to the BRAF inhibitor dabrafenib and were experiencingdisease progression on therapy before being referred for TIL treatment.Dabrafenib was stopped just prior to TIL therapy and was restartedapproximately 1-2 weeks following TIL to prevent rapid tumour growththat often accompanies dabrafenib discontinuation. Each of these 2patients achieved a qualitative response following TIL (1 durable CR and1 PR). Both patients subsequently discontinued dabrafenib once inresponse following TIL. Because both of these patients had disease thathad become refractory to dabrafenib, it is reasonable to conclude thatthe clinical benefit they experienced following TIL was due to TIL andnot the transient resumption of dabrafenib. Therefore, a sensitivityanalysis of response was performed including these patients asresponders. In this sensitivity analysis, the response rate was 14/21(67%) with 4 (19%) complete responders and 10 (48%) partial responders(Table 22).

TABLE 22 Summary of Best Overall Response, Sensitivity Analysis (AllTreated Subjects) All Treated Subjects (N = 21) Best Overall ResponseComplete Response (CR)  4 (19.0) 95% Cl (Clopper Pearson method) 5.4,41.9 Partial Response (PR) 10 (47.6) 95% Cl (Clopper-Pearson method)25.7, 70.2 Stable Disease (SD)  4 (19.0) 95% Cl (Clopper-Pearson method) 5.4, 41.9 Progressive Disease (PD)  3 (14.3) 95% Cl (Clopper Pearsonmethod) 3.0, 36.3 Response Rate (CR + PR) 14 (66.7) 95% Cl (ClopperPearson method) 43.0, 95.4 Disease Control Rate (CR + PR + SD) 18 (85.7)95% Cl (Clopper Pearson method) 63.7, 97.0

Responses were generally consistent across subgroups by importantbaseline and disease characteristics including age, number of diseasesites, number of prior lines of therapies, prior BRAF inhibitor, priorPD-1 inhibitor, baseline brain metastasis, and baseline tumour burden.Notably, in the 4 patients treated with the manufacturing process mostsimilar to that of ITIL-168, the overall response rate (75%) and the CRrate (25%) were consistent with the broader population. Of the 15patients with quantitative response based on CT and/or MRI scans, 14 haddetailed tumour measurements and the maximum percentages of tumourreduction from baseline were presented in a waterfall plot (FIG. 74 ).One patient had a best overall response of PD but did not have anypost-treatment target lesion measures reported (progression determinedby observation of new lesions) and hence was not presented in the plot.

The median progression-free survival (PFS) time per quantitativeresponses data (N=15) was 6.7 months, with 4 patients having an ongoingresponse (2 CRs and 2 PRs) without any subsequent therapies at the timeof the analysis cutoff. The median PFS time based on both quantitativeand qualitative responses data (N=21) was 6.7 months, with 5 subjectshaving an ongoing response (3 CRs and 2 PRs) without any subsequenttherapies. The median overall survival (OS) time with all 21 treatedpatients was 21.3 months (FIG. 75A). The median OS time of the 15patients with quantitative response data was 16 months (FIG. 75B).However, the median OS time for responders (per quantitative responseonly, N=8) was not reached, whereas the median OS time for nonresponders(N=7) was 6.5 months (FIG. 75C).

Example 14—Genetically Modified TIL

TABLE 23 Reagents and Equipment Reagent Manufacturer Catalog # 15 mLPolypropylene Centrifuge Tubes Appleton Woods AB031 50 mL PolypropyleneCentrifuge Tubes Appleton Woods AB028 Dulbecco′s Phosphate BufferedSaline Sigma-Aldrich D8537-24X500ML Fetal Bovine Serum (Heatinactivated) Sigma-Aldrich F9665-500ML TCM-CT4834/GIBCO CUSTOM P158718Gibco Penicillin-Streptomycin Sigma-Aldrich P0781-100ML TC 6-well plateStarLab CC7682-7506 Sterile 1.5 mL Eppendorf StarLab S1615-5510 Non-TCfat-bottom 96-well plate Falcon 353072 96 well U bottom plate Falcon351177 FACS tube SLS 352063 TC 24-well plate StarLah CC7682-7524Microplate For Suspension Culture, 96 Grenier, Bio-One 655185 Well,F-Bottom T cell TransACT (TM), human Miltenyi 130-111-160 Gentamyeinamphotericin Invitrogen 10184583 (ThermoFisher Scientific) Prolcukin(Aldesleukin) IL-2 Novartis PL-00101/0936 Heraeus Megafuge 40R,Refrigerated Thermo Scientific 75004518 Centrifuge IncuSafe CO2Incubator PHCBI MCO-170AIC-PE NovoCyte 3005 Flow Cytometer SystemAgilent Technologies 2010064D (CE-IVD) NovoExpress Software AgilentTechnologies

Tumor digest cryovials are removed from liquid nitrogen storage andthawed in a 37° C. water bath until the cell suspension is just melted(D1). The cell suspension is removed to a 15 mL falcon, topped up withPBS up to 10 mL, centrifuged at 400 g for 5 min and the supernatantdecanted.

The cell pellet is resuspended in pre warmed appropriate T-cell media,and cell counts are performed to determine viability using Trypan blue.Cells are resuspended at a density of 1×10⁶ cells per mL.

Cells to be cultured without activation are resuspended at 0.5×10⁶ cellsper ml and 2 ml (1×10⁶ cells) are placed in a well of a 24 well tissueculture plate with IL-2 (3000 IU/mL). The cells are cultured in ahumidified 37° C. incubator until transduction with IL-2 (3000 IU/mL)addition every 2-3 days.

For the cells to be transduced on D3 and D4 activation of the cellsoccurs on Dl. For the cells to be transduced on D7 and D8 activation ofthe cells occurs on D5.

For TIL activation, 0.5×10⁶ cells/mL are place in a 24 well tissueculture plate with 3000 IU/mL IL-2. 10 μL of T cell TransACT™ is addedper 1×10⁶ cells of TIL suspension (1:1 ratio) and the cells areincubated for 48 h in a 37° C. incubator

Transduction First Day (D3 or D7)

Collect the cells from the 24 well plate into a 15 mL falcon tube, topup with 10 mL TCM and spin at 400 g for 5 min. Count the cells usingTrypan blue and resuspend at 1×10⁶ cells per mL.

Use 1×10⁵ cells (100 μL) per well in 96 well flat bottom plate are usedfor each transduction method. If transducing in 24 well plate, place1×10⁶ cells per well (500 If transducing in 6 well plate, place 5×10⁶cells per well (2 mL).

Prepare a master mix of lentivirus (MOI5) and IL-2 (3000 IU/mL) byresuspending in TCM to a final of 100 μL per 10⁵ cells per condition (orthe appropriate density and volume for 24 well and 6 well plates).Prepare a mastermix volume for number of wells+1 to account forpipetting losses.

For the NT cells (MOCK) prepare a master mix of TCM and IL-2 (3000IU/mL) per 100 μL in 96 well flat bottom plate. For the 24 well and 6well plates, resuspend the MOCK T cells in 500 μL and 2 mL,respectively, with IL-2 (3000 IU/mL).

Remove the supernatant from the cells in Eppendorf or 15 mL falcon tubesand resuspend cells in the appropriate 100 μL of master mix per 1×10 5cells (or the appropriate density and volume for 24 well and 6 wellplates) depending on the condition.

Resuspend properly each condition and transfer the cells onto a non-TCflat-bottom 96-well, 24 well or 6 well plates, accordingly.

In the 96 well plate transduction add 200 μL PBS to surrounding wells toprevent evaporation.

Incubate cells overnight in a humidified 37° C. incubator.

Transduction Second Day (D4 or D8)

Collect the cells by resuspending up and down from the 96 well flatbottom plates and transfer to a 96 well U bottom plate. (Collection froma 24 well or a 6 well plates is performed in a 15 mL falcon.) Spin theplate at 400 g for 5 min and wash the cells with TCM.

Use 1×10 5 cells (100 μL) per well in 96 well flat bottom plate for eachtransduction method. if transducing in 24 well plate, place 1×10⁶ cellsper well (500 if transducing in 6 well plate, place 5×10⁶ cells per well(2 mL).

Prepare a master mix of lentivirus (MOI5) and IL-2 (3000 IU/mL) byresuspending in TCM to a final of 100 μl per 10⁵ cells per condition (orthe appropriate density and volume for 24 well and 6 well plates).Prepare a mastermix volume for number of wells+1 to account forpipetting losses.

For the NT cells (MOCK) prepare a master mix of TCM and IL-2 (3000IU/mL) per 100 μL for the 96 well flat bottom plate. For the 24 well and6 well plates, resuspend the MOCK T cells in 500 μL and 2 mL,respectively, with IL-2 (3000 IU/mL).

Remove the supernatant from the cells in Eppendorf or falcon tubes andresuspend cells in the appropriate 100 μL of master mix per 1×10 5 cells(or the appropriate density and volume for 24 well and 6 well plates)depending on the condition.

Resuspend properly each condition and transfer the cells onto a non-TCflat-bottom 96-well, 24 well or 6 well plates, accordingly. In the 96well plate transduction add 200 μl, PBS to surrounding wells to preventevaporation. Incubate cells overnight a humidified 37° C. incubator.

The next day transfer the cells into new 96 well round bottom plates, 24well or 6 well plates, in fresh media with IL-2 (3000 IU/mL) andincubate for 72 hrs in a humidified 37° C. incubator.

The final volume for 96 well plate is 200 μL per well; the final volumefor 24 well plate is 2 mL per well; the final volume for 6 well plate is5 mL per well. IL-2 (3000 IU/mL) is added every 2-3 days.

The cells are stained for transduction efficiency on D8 for D3+D4transductions and D12 for D7+D8 transductions.

Outgrowth of TILS

Mock and transduced cells are maintained in 96 well U-bottom platesuntil they are placed into a REP.

For the cell maintenance, every 2-3 days half of the media is removedand replaced with fresh TCM and IL-2 (3000 IU/mL). For a 96 well plateremove and replace 100 μl of media to a final volume of 200 μL. For a 24well plate remove and replace 1 mL of media to a final volume of 2 mL.For a 6 well plate remove and replace 1 mL of media to a final volume of2 mL.

The REP begins on D13 (12 days of outgrowth).

The invention is further described by the following numbered paragraphs:

1. A method for isolating a therapeutic population of cryopreservedunmodified tumor infiltrating lymphocytes (UTIL) comprising: (a)aseptically disaggregating a tumor resected from a subject therebyproducing a disaggregated tumor, wherein the tumor is sufficientlydisaggregated so that the cell suspension can be cryopreserved; (b)cryopreserving the disaggregated tumor the same day as step (a) bycooling or maintaining at a low temperature; (c) optionally storing thecryopreserved disaggregated tumor; (d) performing a first expansion byculturing the disaggregated tumor in a cell culture medium comprisingIL-2 to produce a first population of UTILs; (e) performing a secondexpansion by culturing the first population of UTILs with additionalIL-2, OKT-3, and antigen presenting cells (APCs), to produce a secondpopulation of TILs; and (f) harvesting and/or cryopreserving the secondpopulation of UTILs.

2. The method of paragraph 1, wherein the disaggregation comprisesphysical disaggregation, enzymatic disaggregation, or physical andenzymatic disaggregation.

3. The method of paragraph 1 or 2, wherein the cooling is at acontrolled rate.

4. The method of paragraph 3, wherein controlled rate freezing is about−2° C./minute to about −60° C.

5. The method of any one of paragraphs 1-5, wherein the disaggregatedtumor is cellularized.

6. The method of any one of paragraphs 1-5, wherein the disaggregatedtumor is purified.

7. The method of any one of paragraphs 1-6, wherein a single cellsuspension is provided after step (a).

8. The method of any one of paragraphs 1-7, wherein the first populationof UTILs is about 1-20 million UTILs.

9. The method of any one of paragraphs 1-8, wherein step (d) furthercomprises growth of the UTIL out of the tumor starting material followedby a rapid expansion in step (e).

10. The method of paragraphs 9 wherein step (d) is performed for abouttwo weeks and step (e) is performed for about two weeks.

11. The method of any one of paragraphs 1-10 wherein step (d) and/orstep (e) further comprises adding IL-7, IL-12, IL-15, IL-18, IL-21 or acombination thereof.

12. The method of any one of paragraphs 1-11, further comprising step(g) suspending the second population of UTILs.

13. The method of paragraphs 12, wherein the suspending is in bufferedsaline, human serum albumin and dimethylsulfoxide (DMSO).

14. The method of any one of paragraphs 1-13, wherein step (f) iscryopreserving and further comprising a final step of thawing the UTILs.

15. The method of paragraphs 14, wherein the thawed UTILs are ready forinfusion as a single dose with no further modification.

16. A therapeutic population of cryopreserved unmodified tumorinfiltrating lymphocytes (UTIL) obtained by the method of any one ofparagraphs 1-15.

17. The therapeutic population of paragraphs 16 wherein the populationcomprises about 5×10⁹ to 5×10¹⁰ of T cells.

18. A cryopreserved bag of the therapeutic population of paragraphs 16or 17.

19. The cryopreserved bag of paragraphs 18 for use in intravenousinfusion.

20. A method for treating cancer comprising administering thetherapeutic population of paragraphs 14 or 15 or the cryopreserved bagof paragraphs 18 or 19.

21. The method of paragraphs 20, wherein the cancer is bladder cancer,breast cancer, cancer caused by human papilloma virus, cervical cancer,head and neck cancer (including head and neck squamous cell carcinoma(HNSCC), lung cancer, melanoma, ovarian cancer, non-small-cell lungcancer (NSCLC), renal cancer or renal cell carcinoma.

The invention is further described by the following numbered paragraphs:

1. A method for isolating a therapeutic population of cryopreservedunmodified tumor infiltrating lymphocytes (UTIL) comprising: (a)resecting a tumor from a subject; (b) storing the resected tumor in asingle use aseptic kit, wherein the aseptic kit comprises: adisaggregation module for receipt and processing of material comprisingsolid mammalian tissue; an optional enrichment module for filtration ofdisaggregated solid tissue material and segregation of non-disaggregatedtissue and filtrate; and a stabilization module for optionally furtherprocessing and/or storing disaggregated product material, wherein eachof the modules comprises one or more flexible containers connected byone or more conduits adapted to enable flow of the tissue material therebetween; and wherein each of the modules comprises one or more ports topermit aseptic input of media and/or reagents into the one or moreflexible containers; (c) aseptically disaggregating the resected tumorin the disaggregation module thereby producing a disaggregated tumor,wherein the resected tumor is sufficiently disaggregated if it can becryopreserved without cell damage; (d) cryopreserving the disaggregatedtumor in the stabilization module; (e) performing a first expansion byculturing the disaggregated tumor in a cell culture medium comprisingIL-2 to produce a first population of UTILs; (f) performing a secondexpansion by culturing the first population of UTILs with additionalIL-2, OKT-3, and antigen presenting cells (APCs), to produce a secondpopulation of TILs; (g) harvesting and/or cryopreserving the secondpopulation of UTILs. In some embodiments, step a) is optional.

2. The method of paragraph 1, wherein the disaggregation comprisesphysical disaggregation, enzymatic disaggregation, or physical andenzymatic disaggregation.

3. The method of paragraph 1 or 2, wherein the disaggregated tumor iscellularized.

4. The method of any one of paragraphs 1-3, wherein a single cellsuspension is provided after step (c).

5. The method of any one of paragraphs 1-4, wherein the first populationof UTILs is about 1-20 million UTILs.

6. The method of any one of paragraphs 1-5, wherein step (e) furthercomprises growth of the UTILs out of the resected tumor startingmaterial followed by the rapid expansion of step (f).

7. The method of paragraph 6, wherein step (e) is performed for abouttwo weeks and step (f) is performed for about two weeks.

8. The method of any one of paragraphs 1-7, wherein step (e) and/or step(f) further comprises adding IL-7, IL-12, IOL-15, IL-18, IL-21, or acombination thereof.

9. The method of any one of paragraphs 1-7, further comprising step (h)suspending the second population of UTILs.

10. The method of paragraph 9, wherein the suspending is in bufferedsaline, human serum albumin, and dimethylsulfoxide (DMSO).

11. The method of any one of paragraphs 1-9, wherein step (g) iscryopreserving and further comprising a final step of thawing the UTILs.

12. The method of paragraph 10, wherein the thawed UTILs are ready forinfusion as a single dose with no further modification.

13. A therapeutic population of cryopreserved UTILs obtained by themethod of any one of paragraphs 1-11.

14. The therapeutic population of paragraph 13, wherein the populationcomprises about 5×10⁹ to 5×10¹⁰ of T cells.

15. A cryopreserved bag of the therapeutic population of paragraph 13 or14.

16. The cryopreserved bag of paragraph 15 for use in intravenousinfusion.

17. A method for treating cancer comprising administering thetherapeutic population of paragraph 13 or 14 or the cryopreserved bag ofparagraph 15 or 16.

18. The method of paragraph 17, wherein the cancer is bladder cancer,breast cancer, cancer caused by human papilloma virus, cervical cancer,head and neck cancer (including head and neck squamous cell carcinoma[HNSCC]), lung cancer, melanoma, ovarian cancer, non-small-cell lungcancer (NSCLC), renal cancer or renal cell carcinoma.

19. The method of paragraph 1, wherein the one or more flexiblecontainers of the aseptic kit comprises a resilient deformable material.

20. The method of paragraph 1, wherein the one or more flexiblecontainers of the disaggregation module of the aseptic kit comprises oneor more sealable openings.

21. The method of paragraph 20, wherein the flexible container of thedisaggregation module of the aseptic kit comprises a heat sealable weld.

22. The method of paragraph 1, wherein the one or more flexiblecontainers of the aseptic kit comprises internally rounded edges.

23. The method of paragraph 1, wherein the one or more flexiblecontainers of the disaggregation module of the aseptic kit comprisesdisaggregation surfaces adapted to mechanically crush and shear thesolid tissue therein.

24. The method of paragraph 1, wherein the one or more flexiblecontainers of the enrichment module of the aseptic kit comprises afilter that retains a retentate of cellularized disaggregated solidtissue.

25. The method of paragraph 1, wherein the one or more flexiblecontainers of the stabilization module of the aseptic kit comprisesmedia formulation for storage of viable cells in solution or in acryopreserved state.

26. The method of paragraph 1, wherein the aseptic kit further comprisesa digital, electronic, or electromagnetic tag identifier.

27. The method of paragraph 26, wherein the tag identifier of theaseptic kit relates to a specific program that defines: a type ofdisaggregation and/or enrichment and/or stabilization process; one ormore types of media used in said processes; including and optionalfreezing solution suitable for controlled rate freezing.

28. The method of paragraph 1, wherein the same flexible container canform part of one or more of the disaggregation module, the stabilizationmodule, and the optional enrichment modules.

29. The method of paragraph 1, wherein the disaggregation module of theaseptic kit comprises a first flexible container for receipt of thetissue to be processed.

30. The method of paragraph 1, wherein the disaggregation module of theaseptic kit comprises a second flexible container comprising the mediafor disaggregation.

31. The method of paragraph 1, wherein the optional enrichment module ofthe aseptic kit comprises the first flexible container and a thirdflexible container for receiving the enriched filtrate.

32. The method of paragraph 1, wherein both the disaggregation moduleand the stabilization module of the aseptic kit comprise the secondflexible container and wherein the second container comprises digestionmedia and stabilization media.

33. The method of paragraph 1, wherein the stabilization module of theaseptic kit comprises a fourth flexible container comprisingstabilization media.

34. The method of paragraph 1, wherein the stabilization module of theaseptic kit also comprises the first flexible container and/or thirdflexible container for storing and/or undergoing cryopreservation.

35. A method for isolating a therapeutic population of cryopreservedunmodified tumor infiltrating lymphocytes (UTIL) comprising: (a)resecting a tumor from a subject; (b) storing the resected tumor in anautomated device for semi-automated aseptic disaggregation and/orenrichment and/or stabilization of cells or cell aggregates frommammalian solid tissue comprising a programmable processor and a singleuse aseptic kit, wherein the aseptic kit comprises: a disaggregationmodule for receipt and processing of material comprising solid mammaliantissue; an optional enrichment module for filtration of disaggregatedsolid tissue material and segregation of non-disaggregated tissue andfiltrate; and a stabilization module for optionally further processingand/or storing disaggregated product material, wherein each of themodules comprises one or more flexible containers connected by one ormore conduits adapted to enable flow of the tissue material therebetween; and wherein each of the modules comprises one or more ports topermit aseptic input of media and/or reagents into the one or moreflexible containers; (c) aseptically disaggregating the resected tumorthereby producing a disaggregated tumor, wherein the resected tumor issufficiently disaggregated if it can be cryopreserved without celldamage; (d) cryopreserving the disaggregated tumor in the stabilizationmodule; (e) performing a first expansion by culturing the disaggregatedtumor in a cell culture medium comprising IL-2 to produce a firstpopulation of UTILs; (f) performing a second expansion by culturing thefirst population of UTILs with additional IL-2, O KT-3, and antigenpresenting cells (APCs), to produce a second population of TILs; (g)harvesting and/or cryopreserving the second population of UTILs. In someembodiments, step a) is optional.

36. The method of paragraph 35, wherein the automated device furthercomprises a radio frequency identification tag reader for recognition ofthe aseptic kit.

37. The method of paragraph 36, wherein the programmable processor ofthe automated device is capable of recognizing the aseptic kit via thetag and subsequently executes the kit program defining the type ofdisaggregation, enrichment, and stabilization processes, and therespective media types required for said processes.

38. The method of paragraph 35, wherein the programmable processor ofthe automated device is adapted to communicate with and control one ormore of: the disaggregation module; the enrichment module; and thestabilization module.

39. The method of paragraph 38, wherein the programmable processor ofthe automated device controls the disaggregation module to enable aphysical and/or biological breakdown of the solid tissue material.

40. The method of paragraph 39, wherein the programmable processorcontrols the disaggregation module to enable a physical and enzymaticbreakdown of the solid tissue material.

41. The method of paragraph 40, wherein the enzymatic breakdown of thesolid tissue material is by one or more media enzyme solutions selectedfrom the group consisting of collagenase, trypsin, lipase,hyaluronidase, deoxyribonuclease, Liberase HI, pepsin, and mixturesthereof.

42. The method of paragraph 35, wherein the programmable processorcontrols disaggregation surfaces within the disaggregation flexiblecontainers that mechanically crush and shear the solid tissue,optionally wherein the disaggregation surfaces are mechanical pistons.

43. The method of paragraph 35, wherein the programmable processorcontrols the stabilization module to cryopreserve the enricheddisaggregated solid tissue in the container, optionally using aprogrammable temperature.

44. The method of paragraph 35, wherein the automated device furthercomprises one or more of, in any combination: sensors capable ofrecognizing whether a disaggregation process has been completed in thedisaggregation module prior to transfer of the disaggregated solidtissue to the optional enrichment module; weight sensors to determine anamount of media required in the containers of one or more of thedisaggregation module; the enrichment module; and/or the stabilizationmodule and control the transfer of material between respectivecontainers; sensors to control temperature within the containers of theone or more of the disaggregation module; the enrichment module; and/orthe stabilization module; at least one bubble sensor to control transferof media between the input and output ports of each container in themodule; at least one pump, optionally a peristaltic pump, to controltransfer of media between the input and output ports; pressure sensorsto assess the pressure within the enrichment module; one or more valvesto control a tangential flow filtration process within the enrichmentmodule; and/or one or more clamps to control the transfer of mediabetween the input and output ports of each module.

45. The method of paragraph 35, wherein the programmable processor ofthe automated device is adapted to maintain an optimal storagetemperature range in the stabilization module until the container isremoved; or executes a controlled freezing step.

46. The method of paragraph 35, wherein the automated device furthercomprises a user interface.

47. The method of paragraph 46, wherein the interface comprises adisplay screen to display instructions that guide a user to inputparameters, confirm pre-programmed steps, warn of errors, orcombinations thereof.

48. The method of paragraph 35, wherein the automated device is adaptedto be transportable.

49. A semi-automatic aseptic tissue processing method for isolating atherapeutic population of UTILs comprising the steps of: (a)automatically determining aseptic disaggregation tissue processing stepsand their associated conditions from a digital, electronic, orelectromagnetic tag identifier associated with an aseptic processingkit, wherein the aseptic kit comprises: a disaggregation module forreceipt and processing of material comprising solid mammalian tissue; anoptional enrichment module for filtration of disaggregated solid tissuematerial and segregation of non-disaggregated tissue and filtrate; and astabilization module for optionally further processing and/or storingdisaggregated product material, wherein each of the modules comprisesone or more flexible containers connected by one or more conduitsadapted to enable flow of the tissue material there between; and whereineach of the modules comprises one or more ports to permit aseptic inputof media and/or reagents into the one or more flexible containers; (b)resecting a tumor from a subject; (c) placing the tumor into theflexible plastic container of the disaggregation module of the aseptickit; (d) processing the tumor by automatically executing the one or moretissue processing steps by communicating with and controlling: thedisaggregation module; wherein the resected tumor is asepticallydisaggregated thereby producing a disaggregated tumor, wherein theresected tumor is sufficiently disaggregated if it can be cryopreservedwithout cell damage; the optional enrichment module wherein thedisaggregated tumor is filtered to remove disaggregated solid tissuematerial and to segregate non-disaggregated tissue and filtrate; thestabilization module wherein the disaggregated tumor is cryopreserved;(e) performing a first expansion by culturing the disaggregated tumor ina cell culture medium comprising IL-2 to produce a first population ofUTILs; (f) performing a second expansion by culturing the firstpopulation of UTILs with additional IL-2, OKT-3, and antigen presentingcells (APCs), to produce a second population of TILs; and (g) harvestingand/or cryopreserving the second population of UTILs.

The invention is further described by the following numbered paragraphs:

1. A flexible container for processing tissue comprising: one or morelayers made of a sealable polymer, wherein at least three edges of theflexible container are sealed during manufacturing; an open edge on theflexible container through which tissue material is inserted during use;and one or more connectors configured to couple the flexible containerto at least one element through tubing; wherein a section proximate theopen edge is sealed after tissue material is positioned within theflexible container to form a seal.

2. The flexible container of paragraph 1 wherein the seal comprises atleast a three mm wide area parallel to the open edge and spaced awayfrom the open edge of the flexible container.

3. The flexible container of paragraph 1 further comprises a clamphaving protrusions and positioned proximate the seal and spaced furtherfrom the open edge of the flexible container than the seal.

4. The flexible container of paragraph 3 wherein during use acombination of the seal and the clamp is configured to withstand a 100 Nforce applied to the flexible container.

5. The flexible container of paragraph 3 wherein during use acombination of the seal and the clamp is configured to withstand a 75 Nforce applied to the flexible container.

6. The flexible container of paragraph 1 wherein the seal comprises atleast a five mm wide area parallel to the open edge and spaced away fromthe open edge of the flexible container.

7. The flexible container of paragraph 1 wherein the flexible containeris used for disaggregation of the tissue material.

8. The flexible container of paragraph 1, wherein the flexible containeris used for disaggregation of the tissue material, filtration ofdisaggregated tissue material, and segregation of non-disaggregatedtissue and filtrate.

9. The flexible container of paragraph 1, further comprising a resilientdeformable material.

10. The flexible container of paragraph 1, further comprising one ormore indicators.

11. The flexible container of paragraph 1, further comprising one ormore marks.

12. The flexible container of paragraph 1 wherein the seal is formedusing a heat sealer operating at a predetermined pressure, apredetermined temperature, and predetermined time frame.

13. The flexible container of paragraph 1 wherein the flexible containeris configured to be used with a device that mechanically crushes tissuematerial placed in the flexible container.

14. The flexible container of paragraph 1 wherein the flexible containeris configured to shear the tissue material.

15. Use of the flexible container according to paragraph 1 in asemi-automated or an automated process for the aseptic disaggregation,stabilization and optional enrichment of mammalian cells or cellaggregates.

16. A system for extraction of a desired material from tissuecomprising: a kit comprising: a disaggregation flexible container; astabilization flexible container; and at least one indicator tagpositioned on at least one of the disaggregation flexible container orthe stabilization flexible container capable of providing at least oneof a source of tissue, a status of the tissue, or an identifier; adisaggregation element capable of treating at least some tissue in adisaggregation flexible container to form a processed fluid; anenrichment element capable of enriching at least some of the processedfluid to form the desired material; a stabilization element capable ofstoring a portion of the desired material in the stabilization flexiblecontainer; and at least one indicator tag reader positioned on at leastone of the disaggregation element or the stabilization element capableof providing at least one of a source of tissue, or a status of thetissue at the stabilization element.

17. The system of paragraph 15 wherein the desired material comprisestumor infiltrating lymphocytes (TILs).

18. The system of paragraph 15 wherein one or more types of media areused in the processes by the disaggregation element and thestabilization element.

19. The system of paragraph 15 further comprising a cryopreservationmedia for use in the stabilization element capable of controlled ratefreezing.

20. The system of paragraph 15 wherein the disaggregation flexiblecontainer comprises a disaggregation bag having an open edge which issealed during use and the stabilization flexible container is astabilization bag.

21. An automated device for semi-automated aseptic disaggregation and/orenrichment and/or stabilization of cells or cell aggregates frommammalian solid tissue comprising: a programmable processor; and a kitcomprising at least one of the flexible container of any of paragraphs 1to 15 as a disaggregation flexible container.

22. The automated device of paragraph 21, further comprising anindicator tag reader.

23. The automated device of paragraph 21, further comprising a radiofrequency identification tag reader to recognize a component of the kit.

24. The automated device of paragraph 21, wherein the programmableprocessor is capable of recognizing the component of the kit via the tagand subsequently executes a program defining the type of disaggregation,enrichment and stabilization processes and the respective media typesrequired for those processes.

25. The automated device of paragraph 21 wherein the programmableprocessor controls a disaggregation element of the automated device toenable a physical and/or biological breakdown of the solid tissue in thedisaggregation flexible container.

26. The automated device of paragraph 25 wherein the programmableprocessor controls a disaggregation surface proximate the disaggregationflexible container which mechanically crushes and shears the solidtissue positioned in the disaggregation flexible container, optionallywherein the disaggregation surfaces are mechanical pistons.

27. The automated device of paragraph 21 wherein the programmableprocessor controls a disaggregation element of the automated device toenable a physical and enzymatic breakdown of the solid tissue in thedisaggregation flexible container.

28. The automated device of paragraph 27 wherein the enzymatic breakdownof the solid tissue is by one or more media enzyme solutions selectedfrom collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease,Liberase HI, pepsin, or mixtures thereof.

29. The automated device of paragraph 21 wherein the device comprises atleast two of a disaggregation element; an enrichment element; and astabilization element; and wherein the programmable processor is adaptedto communicate with and control one or more of: the disaggregationelement; the enrichment element; and the stabilization element.

30. The automated device of any one of paragraphs 29 wherein theprogrammable processor controls the stabilization element tocryopreserve the enriched disaggregated solid tissue in thecryopreservation container, optionally using a programmable temperature.

31. The automated device of any one of paragraphs 29 wherein the devicefurther comprises one or more of the additional components in anycombination: sensors capable of recognizing whether a disaggregationprocess has been completed in the disaggregation element prior totransfer of the disaggregated solid tissue to the optional enrichmentelement; weight sensors to determine an amount of media required in thecontainers of one or more of the disaggregation element; the enrichmentelement; and/or the stabilization element and control the transfer ofmaterial between respective containers; sensors to control temperaturewithin the containers of the one or more of the disaggregation element;the enrichment element; and/or the stabilization element; at least onebubble sensor to control the transfer of media between the input andoutput ports of each container in the element; at least one pump,optionally a peristaltic pump, to control the transfer of media betweenthe input and output ports; pressure sensors to assess the pressurewithin the enrichment element; one or more valves to control atangential flow filtration process within the enrichment element; and/orone or more clamps to control the transfer of media between the inputand output ports of each element.

32. The automated device of paragraph 29 wherein the programmableprocessor is adapted to maintain an optimal storage temperature range inthe stabilization element until the container is removed; or executes acontrolled freezing step.

33. The automated device of any preceding paragraph, further comprisinga user interface.

34. The automated device of paragraph 26, wherein the interfacecomprises a display screen to display instructions that guide a user toinput parameters, confirm pre-programmed steps, warn of errors, orcombinations thereof.

35. The automated device of paragraph 21 wherein the automated device isadapted to be transportable.

36. An automatic tissue processing method comprising: automaticallydetermining conditions for processing steps and their associatedconditions from a digital, electronic or electromagnetic tag indicatorassociated with a kit; placing a tissue sample into a flexible containerof the kit; and

sealing at least one edge of the flexible container; processing thetissue sample by automatically executing one or more tissue processingsteps by communicating with the indicator and controlling the flexiblecontainer; and filtering at least a portion of the processed tissuesample to generate a filtered fluid; and providing at least some of thefiltered fluid to a cyropreservation flexible container.

37. The method of paragraph 31 wherein processing comprises agitation,extraction, and enzymatic digestion of at least a portion of the tissuesample in the flexible container.

38. The method of paragraph 31 wherein processing comprises agitation,extraction, and enzymatic digestion of at least a portion of the tissuesample in the flexible container and resulting in the extraction of adesired material.

39. The method of paragraph 31 wherein processing comprises agitation,extraction, and enzymatic digestion of at least a portion of the tissuesample in the flexible container and resulting in the extraction oftumor infiltrating lymphocytes (TILs).

40. The method of paragraph 31 wherein the flexible container comprisesheat-sealable material.

41. The method of paragraph 31 wherein the flexible container comprisesat least one of EVA, a vinyl acetate and polyolefin polymer blend, orpolyamide.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A method of identifying potent geneticallymodified T-cells in a population of TILs, comprising: in an isolated andex vivo expanded population of TILs derived from a subject's cancertissue, identifying the presence of genetically modified T-cellsexpressing a combination of markers, the combination comprising each of:CD107a; CD3; IFN-γ; and, a marker for identifying a genetic modificationof the T-cells; wherein, a genetically modified T-cell is determined tobe potent by the presence of the combination of markers.
 2. The methodof claim 1, wherein the genetic modification is transduction with avector.
 3. The method of claim 1, wherein the isolated and ex vivoexpanded population of TILs has undergone one or more processes selectedfrom the group consisting of disaggregation, cryopreservation, thawing,incubation, washing, isoform enrichment, activation with an anti-CD3antibody, co-culturing with an inactivated feeder cell, and co-culturingwith an activator cell.
 4. The method of claim 1, wherein the isolatedand ex vivo expanded population of TILs has undergone ex vivo expansioncomprising outgrowth and rapid expansion.
 5. The method of claim 1,wherein the identifying comprises intracellular staining and flowcytometry.
 6. A method of determining potency of a population of TILs,comprising: in an isolated and ex vivo expanded population of TILsderived from a subject's cancer tissue, quantifying genetically modifiedT-cells expressing a combination of markers, the combination comprisingeach of: a T-cell expressing CD107a; a T-cell expressing CD3; a T-cellexpressing IFN-γ; and, a T-cell expressing a marker for identifying agenetic modification in the T-cell; wherein, a genetically modifiedT-cell is determined to be potent by the presence of the combination ofmarkers; quantifying the total number of genetically modified T-cells inthe expanded population of TILs; and, determining the percent potency ofthe expanded population of TILs by the ratio of genetically modifiedT-cells expressing the combination of markers to the total number ofT-cells in the expanded population of TILs.
 7. The method of claim 6,wherein the genetic modification is transduction with a vector.
 8. Themethod of claim 6, wherein the isolated and ex vivo expanded populationof TILs has undergone one or more processes selected from the groupconsisting of disaggregation, cryopreservation, thawing, incubation,washing, isoform enrichment, activation with an anti-CD3 antibody,co-culturing with an inactivated feeder cell, and co-culturing with anactivator cell.
 9. The method of claim 6, wherein the isolated and exvivo expanded population of TILs has undergone ex vivo expansioncomprising outgrowth and rapid expansion.
 10. The method of claim 6,wherein the identifying comprises intracellular staining and flowcytometry.
 11. A method for treating cancer in a subject comprisingidentifying potent genetically modified T-cells in a population of TILs,comprising: in an isolated and ex vivo expanded population of TILsderived from a subject's cancer tissue, identifying the presence ofgenetically modified T-cells expressing a combination of markers, thecombination comprising each of: CD107a; CD3; IFN-γ; and, a marker foridentifying a genetic modification of the T-cells, wherein, agenetically modified T-cell is determined to be potent by the presenceof the combination of markers, and administering to the subject theidentified potent genetically modified T-cells.
 12. The method of claim11, wherein the genetic modification is transduction with a vector. 13.The method of claim 11, wherein the isolated and ex vivo expandedpopulation of TILs has undergone one or more processes selected from thegroup consisting of disaggregation, cryopreservation, thawing,incubation, washing, isoform enrichment, activation with an anti-CD3antibody, co-culturing with an inactivated feeder cell, and co-culturingwith an activator cell.